Compositions and methods for increasing nematode resistance in plants

ABSTRACT

The invention relates to methods and compositions for increasing resistance or tolerance to a nematode plant pest in a plant or part thereof. Nucleotide sequences that confer resistance or tolerance to nematode plant pests when expressed in a plant are provided as well as compositions comprising the polypeptides encoded by the nucleotide sequences, and transgenic plants and parts thereof comprising the nucleotide sequences.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/974,815 filed on May 9, 2018, which is a division of U.S. patentapplication Ser. No. 14/359,045 filed on May 16, 2014, now U.S. Pat. No.10,000,768, which is a National Phase of PCT Patent Application No.PCT/US2012/065959, having International Filing Date of Nov. 20, 2012,which claims benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application No. 61/562,060 filed on Nov. 21, 2011 andU.S. Provisional Patent Application No. 61/684,234 filed on Aug. 17,2012, the contents of which are incorporated herein by reference intheir entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 85136.txt, created on Dec. 2, 2020, comprising3,013,855 bytes, submitted concurrently with the filing of thisapplication is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to compositions and methods for control ofnematode pests in plants.

BACKGROUND OF THE INVENTION

Nematodes are elongated symmetrical roundworms that constitute one ofthe largest and most successful phyla in the animal kingdom. Manynematode species are free-living and feed on bacteria, whereas othershave evolved into pests or parasites of plants and animals, includinghumans.

Nematode pests of plants are responsible for many billions of dollars ineconomic losses annually. Nematode plant pests feed on stems, buds,leaves and, in particular, on roots of more than 2,000 vegetables,fruits, and ornamental plants, causing an estimated $100-125 billioncrop loss worldwide. Nematodes are present throughout the United States(US), but are mostly a problem in warm, humid areas of the south andwest, as well as in sandy soils. The most economically damaging plantnematode pest genera belong to the family Heterderidae of the orderTylenchida, and include the cyst nematodes [genera Heterodera andGlobodera, e.g., soybean cyst nematode (Heterodera glycines, SCN) andpotato cyst nematodes (G. pallida and G. rostochiensis)], and theroot-knot nematodes (genus Meloidogyne).

Root-knot nematodes infest thousands of different plant speciesincluding vegetables, fruits, and row crops. Cyst nematodes are known toinfest tobacco, cereals, sugar beets, potato, rice, corn, soybeans andmany other crops. Heterodera schachtii (BCN) principally attacks sugarbeets, and Heterodera avenae is a pest of cereals. Heterodera zeae feedson corn, and Globodera rostochiensis and G. pallida feed on potatoes.The soybean cyst nematode (SCN) is present in every soybean-producingstate in the US, and causes total soybean yield losses estimated to benearly $1 billion per year. Once SCN is present in a field, it cannotfeasibly be eradicated using known methods. Although soybean is themajor economic crop attacked by SCN, SCN attacks some fifty hosts intotal, including field crops, vegetables, ornamentals, and weeds.

Cotton root knot nematode (RKN) is a destructive nematode, which formsgalls on the roots of cotton plants. The causative agent is Meloidogyneincognita (Kofoid and White) Chitwood, a nematode which can infest avariety of plant species. Nutrient and water uptake are decreased ininfested plants, and plants may become susceptible to pathogens,especially Fusarium wilt. Consequently, yield is decreased in plantsinfested with RKN. In the US alone, an estimated 10.93% of cotton yieldloss in 2004 was attributed to RKN (Blasingame and Patel, Proceedings ofthe Beltwide Cotton Conferences 1:259-262 (2005). RKN is wide-spreadthroughout the U.S. Cotton Belt. Methods to mitigate RKN damage includerotating cotton crops with non-susceptible crops and application ofcostly nematicides. However, the most effective way for cotton growersto reduce yield loss and crop damage due to RKN is to grow RKN resistantcotton cultivars.

Signs of nematode damage include stunting and yellowing of leaves, aswell as wilting of the plants during hot periods. However, nematodes,including SCN, can cause significant yield loss without obviousabove-ground symptoms. For example, an infestation of SCN to a plant canresult in dwarfed or stunted roots, decrease the number ofnitrogen-fixing nodules on the roots, and/or make the roots moresusceptible to attack by other soil-borne plant pests or pathogens.

In contrast to many viral and bacterial pathogens, little is known aboutthe molecular basis of the nematode-plant interaction, limiting theavailable approaches useful in controlling nematodes. Chemicals usefulin controlling nematode plant pests include organophosphates andcarbamates, the oldest extant class of nematicides, which targetacetylcholinesterase. Imidazole derivatives such as benzimidazole exerttheir nematicidal effects by binding tubulin. Levamisole acts as anagonist on the nicotinic acetylcholine receptor, and avermectins act asirreversible agonists at glutamate-gated chloride channels.Unfortunately, there are certain debilitating nematode infestationswhich are difficult, if not impossible, to eradicate with existingcontrol measures. In addition, the currently available nematode controlagents have drawbacks in terms of efficacy, expense and environmentalsafety. For example, methyl bromide, which is an effective pre-plantsoil fumigant used to control nematodes in many high-input, high-valuecrops in the US, is being phased out due to environmental and humanhealth concerns. However, because methyl bromide has provided a reliablereturn on investment for nematode control, many growers of high valuecrops may be negatively impacted if effective and economicalalternatives are not identified. In addition, environmental concerns,primarily groundwater contamination, ozone depletion, and pesticideresidues in food have prompted the removal of Aldicarb, DGBCP, and othertoxic nematicides from the market by the US Environmental ProtectionAgency. Physical control measures (such as solarization and hot watertreatment), biological control measures (e.g., crop rotation), andintegrated approaches have been used to ameliorate the damage caused byplant nematode pests, but no single method or combination of measures isuniformly effective.

Nematode resistant germplasm and transgenic plants have also beenconsidered as alternatives or complements to chemical control measures.For example, transgenic plants expressing a protease inhibitor showssome resistance to cyst and root-knot nematodes (Urwin et al. 1997.Plant J. 12:455-461). Use of such alternative control measures requiresa greater knowledge of the nematode-plant interaction to achievesatisfactory results. Several studies have generated gene expressiondata suggesting that many host plant genes are up- or down-regulated inresponse to nematode invasion (Szakasits et al. 2009. Plant J.57:771-784; Puthoff et al. 2003. Plant J. 33:911-921; Bethke et al.2009. Proc. Natl. Acad. Sci. 106:8067-8072; Stepanova et al. 2007. PlantCell 19:2169-2185 and Kilian et al. 2007. Plant J. 50:347-363). However,none of these studies aid the skilled person in predicting which, ifany, such genes could be successfully utilized in controlling nematodes,particularly in chimeric gene constructs for deployment in a transgenicplant.

Accordingly, the invention overcomes the deficiencies in the art byproviding compositions and methods comprising recombinant nucleic acidmolecules and their encoded polypeptides for control of nematode pestinfestations in plants.

SUMMARY OF THE INVENTION

The needs outlined above are met by the invention which, in variousembodiments, provides new compositions and methods of controllingeconomically important nematode pests. In particular, transgenic plantsand/or plant parts expressing at least one recombinant nucleic acidmolecule of the invention which modulates expression of proteins of theinvention are found to reduce the ability of nematode pests to survive,grow and reproduce, or of limiting nematode-related damage or loss tothe transgenic plants. The invention is also drawn to transgenicnematode-resistant plants which overexpress or have reduced expressionof a protein of the invention in the transgenic plant and to methods ofusing the transgenic plants alone or in combination with other nematodecontrol measures to confer maximal nematode control efficiency withreduced environmental impact. Transgenic plants and plant parts thathave a protein of the invention overexpressed or inhibited (e.g.,reduced amount and/or reduced activity, and the like, as compared to acontrol) are more tolerant or resistant to nematode pest infestation.For example, the economically important nematode pest, soybean cystnematode (Heterodera glycines) can be controlled by transgenic soybeanplants which over-express a protein of the invention or which comprise anucleic acid molecule of the invention that reduces the expression of aprotein of the invention.

In one aspect of the invention, a method of controlling a nematode plantpest is provided, the method comprising contacting the nematode pestwith a transgenic plant, or part thereof, having incorporated into itsgenome a recombinant nucleic acid molecule that modulates the expressionof one or more polypeptides having the amino acid sequence of SEQ IDNOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046,or any combination thereof, thereby controlling the nematode plant pest.In another aspect of the invention, the recombinant nucleic acidmolecule is capable of producing a double stranded RNA comprising anantisense strand and a sense strand, wherein the antisense strand iscomplementary to a portion of a nucleotide sequence encoding the one ormore polypeptides, the portion comprising, consisting essentially of,consisting of about 18 to about 25 consecutive nucleotides havingsubstantial identity to any one of the nucleotide sequences of SEQ IDNOs:1-28, SEQ ID NOs:43-134, SEQ ID NOs:210-242, SEQ ID NOs:261-664, orany combination thereof.

In yet another aspect of the invention, a recombinant nucleic acidmolecule is provided, the nucleic acid molecule comprising a nucleotidesequence operatively linked to a promoter that functions in a plant orplant cell, wherein the nucleotide sequence is: (a) a nucleotidesequence of any of SEQ ID NOs:1-28, SEQ ID NOs:43-134, SEQ IDNOs:210-242, SEQ ID NOs:261-664; (b) a nucleotide sequence that encodesa polypeptide comprising the amino acid sequence of any of SEQ IDNOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046;(c) a nucleotide sequence having at least 70% sequence identity to anucleotide sequence of (a) and (b) above; (d) a nucleotide sequencewhich anneals under stringent hybridization conditions to the nucleotidesequence of (a), (b) or (c); (e) a nucleotide sequence that differs fromthe nucleotide sequences of (a), (b), (c) or (d) above due to thedegeneracy of the genetic code; or (f) any combination of the nucleotidesequences of (a)-(e).

In another aspect of the invention, a polypeptide is provided, thepolypeptide comprising, consisting essentially of, or consisting of anamino acid sequence of any of SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQID NOs:243-260, SEQ ID NOs:665-1046, or any combination thereof.

In a further aspect of the invention, a method of producing a transgenicplant cell, comprising introducing into a plant cell a recombinantnucleic acid molecule is provided, the recombinant nucleic acid moleculecomprising a nucleotide sequence operatively linked to a promoter thatfunctions in a plant or plant cell, wherein the nucleotide sequence is:(a) a nucleotide sequence of any of SEQ ID NOs:1-28, SEQ ID NOs:43-134,SEQ ID NOs:210-242, SEQ ID NOs:261-644; (b) a nucleotide sequence thatencodes a polypeptide comprising the amino acid sequence of any one ofthe amino acid sequences of SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ IDNOs:243-260, SEQ ID NOs:665-1046; (c) a nucleotide sequence having atleast 70% sequence identity to a nucleotide sequence of (a) and (b)above; (d) a nucleotide sequence which anneals under stringenthybridization conditions to the nucleotide sequence of (a), (b) or (c);(e) a nucleotide sequence that differs from the nucleotide sequences of(a), (b), (c) or (d) above due to the degeneracy of the genetic code; or(f) any combination of the nucleotide sequences of (a)-(e), therebyproducing a transgenic plant cell that can regenerate a plant havingincreased resistance to a nematode plant pest.

A still further aspect of this invention provides a method of producinga soybean plant having increased resistance to infestation by a nematodeplant pest, the method comprising the steps of (a) crossing thetransgenic plant of the invention with itself or another plant toproduce seed comprising the nucleic acid molecule of this invention, orthe vector of the invention; (b) growing a progeny plant from said seedto produce a plant having increased resistance to infestation bynematode plant pests.

In additional aspects of the invention, transgenic plant cells,transgenic plants and parts thereof comprising a nucleic acid moleculethat comprises one or more of the nucleotide sequences of the inventionare provided and methods of using the same to control, suppress, and/orreduce infectivity of a nematode plant pest. Further provided arepolypeptides of the invention and methods of using the same to control,suppress, and/or reduce the infectivity, infestation and/or cystdevelopment of a nematode plant pest, comprising contacting a nematodeplant pest with an effective amount of the polypeptide(s). In someembodiments, contacting the nematode plant pest with an effective amountof a polypeptide comprises contacting the nematode plant pest with atransgenic plant comprising a nucleic acid molecule of the invention.

The invention additionally provides a crop comprising a plurality of thetransgenic plants of the invention planted together in an agriculturalfield. In some aspects, the invention provides a method of improving theyield of a plant crop contacted with a nematode plant pest, the methodcomprising cultivating a plurality of plants comprising a nucleic acidmolecule of the invention as the plant crop, wherein the plurality ofplants of said plant crop have increased resistance to nematodeinfection, thereby improving the yield of said plant crop.

The invention further provides a method of improving yield in a cropcontacted with a nematode plant pest, the method comprising contactingthe nematode plant pest with an effective amount of the polypeptide ofthe invention or the nematicidal composition of the invention, whereinthe yield of the crop is improved.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a binary vector useful for transforming plants and/or plantcells with a recombinant nucleic acid molecule of the invention.

FIG. 2 is an empty vector useful as a negative control in plant and/orplant cell transformation experiments.

FIG. 3 is a binary vector useful for transforming plants and/or plantcells with a recombinant nucleic acid molecule of the invention.

FIG. 4 is an empty vector useful as a negative control in plant and/orplant cell transformation experiments.

FIG. 5 is a schematic illustration of the modified pGI binary plasmidcontaining the At6669 promoter and the GUSintron (pQYN 6669) that can beused for expressing the isolated polynucleotide sequences of theinvention. RB—T-DNA right border; LB—T-DNA left border; MCS—Multiplecloning site; RE—any restriction enzyme; NOS pro=nopaline synthasepromoter; NPT-II=neomycin phosphotransferase gene; NOS ter=nopalinesynthase terminator; Poly-A signal (polyadenylation signal);GUSintron—the GUS reporter gene (coding sequence and intron). In someembodiments, the isolated polynucleotide sequences of the invention werecloned into the vector while replacing the GUSintron reporter gene.

FIG. 6 is a schematic illustration of the modified pGI binary plasmidcontaining the At6669 promoter (pQFN or pQFNc) used for expressing theisolated polynucleotide sequences of the invention. RB—T-DNA rightborder; LB—T-DNA left border; MCS—Multiple cloning site; RE—anyrestriction enzyme; NOS pro=nopaline synthase promoter; NPT-II=neomycinphosphotransferase gene; NOS ter=nopaline synthase terminator; Poly-Asignal (polyadenylation signal); GUSintron—the GUS reporter gene (codingsequence and intron). In some embodiments, the isolated polynucleotidesequences of the invention were cloned into the MCS of the vector.

FIG. 7 is a schematic illustration of pQXNc plasmid, which is a modifiedpGI binary plasmid used for expressing the isolated polynucleotidesequences of some embodiments of the invention. RB—T-DNA right border;LB—T-DNA left border; NOS pro=nopaline synthase promoter;NPT-II=neomycin phosphotransferase gene; NOS ter=nopaline synthaseterminator; RE=any restriction enzyme; Poly-A signal (polyadenylationsignal); 35S—the 35S promoter. In some embodiments, the isolatedpolynucleotide sequences were cloned into the MCS (Multiple cloningsite) of the vector.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NOs:1-14 are nucleotide sequences of the invention comprisinguntranslated regions and coding regions.

SEQ ID NOs:15-28 are coding sequences of the invention encoding theamino acid sequences of SEQ ID NOs:29-42.

SEQ ID NOs:29-42 are amino acid sequences of proteins of the inventionthat when overexpressed or inhibited in transgenic plants confertolerance or resistance to nematodes.

SEQ ID NOs:43-134 are nucleotide sequences that encode the amino acidsequences of SEQ ID NOs:29-42, 135-209.

SEQ ID NOs:135-209 are amino acid sequences of homologues of SEQ IDNOs:29-42.

SEQ ID NOs: 210-223 are nucleotide sequences of the invention comprisinguntranslated regions and coding regions.

SEQ ID NOs:224-242 are coding sequences of the invention encoding theamino acid sequences of SEQ ID NOs:243-260.

SEQ ID NOs: 243-260 are amino acid sequences of proteins of theinvention that when overexpressed or reduced in amount or activity intransgenic plants confer tolerance or resistance to nematodes.

SEQ ID NOs:261-664 are nucleotide sequences of the invention encodinghomologues of the SEQ ID NOs:210-242.

SEQ ID NOs:665-1046 are amino acid sequences of proteins of theinvention that when overexpressed or reduced in amount or activity intransgenic plants confer tolerance or resistance to nematodes.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

This description is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. Thus, theinvention contemplates that in some embodiments of the invention, anyfeature or combination of features set forth herein can be excluded oromitted. In addition, numerous variations and additions to the variousembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention. Hence, the following descriptions are intended toillustrate some particular embodiments of the invention, and not toexhaustively specify all permutations, combinations and variationsthereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art thatthis invention pertains. Further, publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

As used in the description of the embodiments of the invention and theappended claims, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items.

The term “about,” as used herein when referring to a measurable valuesuch as an amount of a compound, dose, time, temperature, and the like,is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1%of the specified amount.

The terms “comprise,” “comprises” and/or “comprising,” when used in thisspecification, specify the presence of stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of (andgrammatical variants) means that the scope of a claim is to beinterpreted to encompass the specified materials or steps recited in theclaim and those that do not materially alter the basic and novelcharacteristic(s)” of the claimed invention. Thus, the term “consistingessentially of” when used in a claim of this invention is not intendedto be interpreted to be equivalent to “comprising.”

The invention is directed in part to the discovery that modulatingexpression by over expressing or reducing expression in a plant of atleast one polypeptide described herein can result in the plant havingincreased resistance to nematode pests. The term “modulating” or“modulates” in the context of the invention means an alteration in theexpression of a protein of the invention by over-expressing the proteinor reducing the expression of the protein. Therefore, in one embodiment,the invention encompasses a method of controlling a nematode plant pestcomprising contacting the nematode pest with a transgenic plant, or partthereof, having incorporated into its genome a recombinant nucleic acidmolecule that modulates the expression of one or more polypeptideshaving any one of the amino acid sequences of SEQ ID NOs:29-42, SEQ IDNOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046, or any combinationthereof, thereby controlling the nematode plant pest. In anotherembodiment, the recombinant nucleic molecule comprises a nucleotidesequence operatively linked to a promoter that functions in a plant orplant cell, wherein the nucleotide sequence comprises, consistsessentially of, or consists of: (a) a nucleotide sequence of any one ofSEQ ID NOs:1-28, SEQ ID NOs:43-134, SEQ ID NOs:210-242, SEQ IDNOs:261-644; (b) a nucleotide sequence that encodes a polypeptidecomprising an amino acid sequence of any one of SEQ ID NOs:29-42, SEQ IDNOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046; (c) a nucleotidesequence having at least 70% sequence identity to the nucleotidesequence of (a) or (b); (d) a nucleotide sequence which anneals understringent hybridization conditions to the nucleotide sequence of (a),(b) or (c), or a complement thereof; (e) a nucleotide sequence thatdiffers from the nucleotide sequences of (a), (b), (c) or (d) above dueto the degeneracy of the genetic code; and (f) any combination of thenucleotide sequences of (a)-(e). In more particular embodiments, thenucleotide sequence can comprise, consist essentially of, or consist of:(a) a nucleotide sequence of any one of SEQ ID NOs:15, 17, 20, 22, 23,24, 26, 226, 227, 228, 230, 232, 233; (b) a nucleotide sequence thatencodes a polypeptide comprising an amino acid sequence of any one ofSEQ ID NOs:29-38, 40-42, 52, 243, 244, 245, 246, 248-252, 254, 256-259;(c) a nucleotide sequence having at least 70% sequence identity to thenucleotide sequence of (a) or (b); (d) a nucleotide sequence whichanneals under stringent hybridization conditions to the nucleotidesequence of (a), (b) or (c), or a complement thereof; (e) a nucleotidesequence that differs from the nucleotide sequences of (a), (b), (c) or(d) above due to the degeneracy of the genetic code; and (f) anycombination of the nucleotide sequences of (a)-(e). In furtherembodiments, the nucleotide sequence can comprise, consist essentiallyof, or consist of: (a) a nucleotide sequence of any one of SEQ ID NOs:56-63, 66-127, 389-401, 408-633, 637-642; (b) a nucleotide sequence thatencodes a polypeptide comprising an amino acid sequence of any one ofSEQ ID NOs: 56-63, 66-127, 389-401, 408-633, 637-642; (c) a nucleotidesequence having at least 70% sequence identity to the nucleotidesequence of (a) or (b); (d) a nucleotide sequence which anneals understringent hybridization conditions to the nucleotide sequence of (a),(b) or (c), or a complement thereof; (e) a nucleotide sequence thatdiffers from the nucleotide sequences of (a), (b), (c) or (d) above dueto the degeneracy of the genetic code; and (f) any combination of thenucleotide sequences of (a)-(e).

In yet another embodiment, the recombinant nucleic acid molecule iscapable of producing a double stranded RNA comprising an antisensestrand and a sense strand, wherein the antisense strand is complementaryto a portion of a nucleotide sequence encoding the one or morepolypeptides, the portion comprising, consisting essentially of,consisting of about 18 to about 25 consecutive nucleotides (e.g., about18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides) havingsubstantial identity to any one of the nucleotide sequences of SEQ IDNOs:1-28, SEQ ID NOs:43-134, SEQ ID NOs:210-242, SEQ ID NOs:261-644, orany combination thereof. In still another embodiment, the recombinantnucleic acid molecule modulates the expression of the one or morepolypeptides of the invention (e.g., SEQ ID NOs:29-42, SEQ IDNOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046) by causingoverexpression of the one or more polypeptides in the transgenic plant.In another embodiment, the recombinant nucleic acid molecule modulatesthe expression of the one or more polypeptides of the invention (e.g.,SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ IDNOs:665-1046) by causing the reduction of or reducing the expression ofthe one or more polypeptides in the transgenic plant.

In another embodiment, the transgenic plant or plant part of theinvention is a transgenic soybean plant, a transgenic sugar beet plant,a transgenic corn plant, a transgenic cotton plant, a transgenic canolaplant, a transgenic wheat plant, a transgenic sugar cane plant, or atransgenic rice plant, or a part thereof.

In still another embodiment, the nematode pest is selected from thegroup consisting of: a cyst nematode (Heterodera spp.), a root knotnematode (Meloidogyne spp.), a lance nematode (Hoplolaimus spp.), astunt nematode (Tylenchorhynchus spp.), a spiral nematode(Helicotylenchus spp.), a lesion nematode (Pratylenchus spp.), a stingnematode (Belonoluimus spp.), a reniform nematode (Rotylenchulusreniformis), a burrowing nematode (Radopholus similis), a ring nematode(Criconema spp.), and any combination thereof. In another embodiment,the nematode is a soybean cyst nematode or a sugar beet cyst nematode.Overexpression or reduced expression of a polypeptide described hereincan result in the plant having increased resistance to nematode plantpests. Thus, in one aspect, the invention provides a recombinant nucleicacid molecule comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, and the like) nucleotide sequences, each ofwhich when expressed in a plant confer increased resistance to anematode plant pest, wherein the one or more nucleotide sequencescomprise, consist essentially of, or consist of: (a) a nucleotidesequence of any of SEQ ID NOs:1-28, SEQ ID NOs:43-134, SEQ IDNOs:210-242, SEQ ID NOs:261-644; (b) a nucleotide sequence that encodesa polypeptide comprising, consisting essentially of, or consisting ofthe amino acid sequence of any of SEQ ID NOs:29-42, SEQ ID NOs:135-209,SEQ ID NOs:243-260, SEQ ID NOs:665-1046; (c) a nucleotide sequencehaving at least 70% sequence identity to a nucleotide sequence of (a)and (b) above; (d) a nucleotide sequence which anneals under stringenthybridization conditions to the nucleotide sequence of (a), (b) or (c);(e) a nucleotide sequence that differs from the nucleotide sequences of(a), (b), (c) or (d) above due to the degeneracy of the genetic code; or(f) any combination of the nucleotide sequences of (a)-(e). In moreparticular embodiments, the nucleotide sequences can comprise, consistessentially of, or consist of: (a) a nucleotide sequence of any of SEQID NOs:15, 17, 20, 22, 23, 24, 26, 226, 227, 228, 230, 232, 233; (b) anucleotide sequence that encodes a polypeptide comprising, consistingessentially of, or consisting of the amino acid sequence of any of SEQID NOs: 29, 31, 34, 36-38, 40, 244-246, 250, 251; (c) a nucleotidesequence having at least 70% sequence identity to a nucleotide sequenceof (a) and (b) above; (d) a nucleotide sequence which anneals understringent hybridization conditions to the nucleotide sequence of (a),(b) or (c); (e) a nucleotide sequence that differs from the nucleotidesequences of (a), (b), (c) or (d) above due to the degeneracy of thegenetic code; or (f) any combination of the nucleotide sequences of(a)-(e). In further embodiments, the nucleotide sequences can comprise,consist essentially of, or consist of: (a) a nucleotide sequence of anyof SEQ ID NOs: 56-63, 66-127, 389-401, 408-633, 637-642; (b) anucleotide sequence that encodes a polypeptide comprising, consistingessentially of, or consisting of the amino acid sequence of any of SEQID NOs: 56-63, 66-127, 389-401, 408-633, 637-642; (c) a nucleotidesequence having at least 70% sequence identity to a nucleotide sequenceof (a) and (b) above; (d) a nucleotide sequence which anneals understringent hybridization conditions to the nucleotide sequence of (a),(b) or (c); (e) a nucleotide sequence that differs from the nucleotidesequences of (a), (b), (c) or (d) above due to the degeneracy of thegenetic code; or (f) any combination of the nucleotide sequences of(a)-(e).

In some embodiments, in addition to the nucleotide sequences describedabove, a nucleic acid molecule of the invention can comprise one or morenucleotide sequences that confer increased resistance to a nematodeplant pest in a plant when expressed in the plant, the one or morenucleotide sequences comprising, consisting essentially of, orconsisting of: a nucleotide sequence of any of SEQ ID NOs:1-28, SEQ IDNOs:43-134, SEQ ID NOs:210-242, SEQ ID NOs:261-644, or any combinationthereof. In some particular embodiments, a nucleic acid molecule of theinvention can comprise one or more nucleotide sequences that conferincreased resistance to a nematode plant pest in a plant when expressedin the plant, the one or more nucleotide sequences comprising,consisting essentially of, or consisting of: a nucleotide sequence ofany of SEQ ID NOs: 15, 17, 20, 22, 23, 24, 26, 226, 227, 228, 230, 232,233, or any combination thereof.

Thus, in some embodiments, the invention provides a recombinant nucleicacid molecule comprising one or more nucleotide sequences, wherein theone or more nucleotide sequences comprise, consist essentially of, orconsist of: (a) a nucleotide sequence of any of SEQ ID NOs:1-28, SEQ IDNOs:43-134, SEQ ID NOs:210-242, SEQ ID NOs:261-644; (b) a nucleotidesequence that encodes a polypeptide comprising, consisting essentiallyof, or consisting of the amino acid sequence of any of SEQ ID NOs:29-42,SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046; (c) anucleotide sequence having significant sequence identity to a nucleotidesequence of (a), (b) above; (d) a nucleotide sequence which annealsunder stringent hybridization conditions to the nucleotide sequence of(a)-(c) above; (e) a nucleotide sequence that differs from thenucleotide sequences of (a)-(d) above due to the degeneracy of thegenetic code; or (f) any combination of the nucleotide sequences of(a)-(e).

In some embodiments of the invention, nucleotide sequences havingsignificant sequence identity to the nucleotide sequences of theinvention are provided. “Significant sequence identity” or “significantsequence similarity” means at least about 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, and/or 100% identity or similarity with another nucleotidesequence. Thus, in additional embodiments, “significant sequenceidentity” or “significant sequence similarity” means a range of about70% to about 100%, about 75% to about 100%, about 80% to about 100%,about 81% to about 100%, about 82% to about 100%, about 83% to about100%, about 84% to about 100%, about 85% to about 100%, about 86% toabout 100%, about 87% to about 100%, about 88% to about 100%, about 89%to about 100%, about 90% to about 100%, about 91% to about 100%, about92% to about 100%, about 93% to about 100%, about 94% to about 100%,about 95% to about 100%, about 96% to about 100%, about 97% to about100%, about 98% to about 100%, and/or about 99% to about 100% identityor similarity with another nucleotide sequence. Therefore, in someembodiments, a nucleotide sequence of the invention is a nucleotidesequence that has significant sequence identity to the nucleotidesequence of any of SEQ ID NOs:1-28, SEQ ID NOs:43-134, SEQ IDNOs:210-242, SEQ ID NOs:261-644. In some particular embodiments, anucleotide sequence of the invention is a nucleotide sequence that hassignificant sequence identity to the nucleotide sequence of any of SEQID NOs:15, 17, 20, 22, 23, 24, 26, 226, 227, 228, 230, 232 and/or 233.

In some embodiments of the invention, the nucleotide sequences and/ornucleic acid molecules of the invention can be expressed to producepolypeptides, each of which when produced in a plant result in increasedresistance to a nematode plant pest. Thus, in other aspects of theinvention, a polypeptide is provided, the polypeptide comprising,consisting essentially of, or consisting of an amino acid sequence ofany of SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ IDNOs:665-1046, wherein production of said polypeptide in a plant resultsin increased resistance to a nematode plant pest in the plant.

A still further aspect of the invention is a nematicidal compositioncomprising one or more polypeptides of the invention. In someembodiments, the composition comprises a polypeptide comprising,consisting essentially of, or consisting of an amino acid sequence ofany of SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ IDNOs:665-1046, or any combination thereof.

In some embodiments, a polypeptide of the invention comprises, consistsessentially of, or consists of an amino acid sequence that is at least70% identical, e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,and/or 100% identical to an amino acid sequence of any of SEQ IDNOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046.

The polypeptides of the invention can be produced in and collected fromcells transformed with the nucleic acid molecules comprising thenucleotide sequences of the invention. Therefore, the polypeptides canbe isolated and provided in a composition of the invention as apartially or fully purified full-length polypeptide, or as an activevariant or fragment thereof, or the polypeptides can be provided as acell extract or cell lysate from the cell or cells of an organismproducing said polypeptide(s). Complete purification is not required inany case. The polypeptide, variant or fragment thereof can be at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%pure (w/w), or more.

In some embodiments, a polypeptide or nucleotide sequence of theinvention can be a conservatively modified variant. As used herein,“conservatively modified variant” refer to polypeptide and nucleotidesequences containing individual substitutions, deletions or additionsthat alter, add or delete a single amino acid or nucleotide or a smallpercentage of amino acids or nucleotides in the sequence, where thealteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.

As used herein, a conservatively modified variant of a polypeptide isbiologically active and therefore possesses the desired activity of thereference polypeptide (e.g., conferring increased resistance to anematode plant pest, reducing the growth of a nematode plant pest,reducing nematode cyst development) as described herein. The variant canresult from, for example, a genetic polymorphism or human manipulation.A biologically active variant of the reference polypeptide can have atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity orsimilarity (e.g., about 40% to about 99% or more sequence identity orsimilarity and any range therein) to the amino acid sequence for thereference polypeptide as determined by sequence alignment programs andparameters described elsewhere herein. An active variant can differ fromthe reference polypeptide sequence by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

Naturally occurring variants may exist within a population. Suchvariants can be identified by using well-known molecular biologytechniques, such as the polymerase chain reaction (PCR), andhybridization as described below. Synthetically derived nucleotidesequences, for example, sequences generated by site-directed mutagenesisor PCR-mediated mutagenesis which still encode a polypeptide of theinvention, are also included as variants. One or more nucleotide oramino acid substitutions, additions, or deletions can be introduced intoa nucleotide or amino acid sequence disclosed herein, such that thesubstitutions, additions, or deletions are introduced into the encodedprotein. The additions (insertions) or deletions (truncations) may bemade at the N-terminal or C-terminal end of the native protein, or atone or more sites in the native protein. Similarly, a substitution ofone or more nucleotides or amino acids may be made at one or more sitesin the native protein.

For example, conservative amino acid substitutions may be made at one ormore predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a protein without altering the biologicalactivity, whereas an “essential” amino acid is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue with a similarside chain. Families of amino acid residues having similar side chainsare known in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Suchsubstitutions would not be made for conserved amino acid residues, orfor amino acid residues residing within a conserved motif, where suchresidues are essential for protein activity.

For example, amino acid sequence variants of the reference polypeptidecan be prepared by mutating the nucleotide sequence encoding the enzyme.The resulting mutants can be expressed recombinantly in plants, andscreened for those that retain biological activity by assaying foractivity against nematodes and plants using standard assay techniques asdescribed herein. Methods for mutagenesis and nucleotide sequencealterations are known in the art. See, e.g., Kunkel (1985) Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.154:367-382; and Techniques in Molecular Biology (Walker & Gaastra eds.,MacMillan Publishing Co. 1983) and the references cited therein; as wellas U.S. Pat. No. 4,873,192. Clearly, the mutations made in the DNAencoding the variant must not disrupt the reading frame and preferablywill not create complimentary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (NationalBiomedical Research Foundation, Washington, D.C.), herein incorporatedby reference.

The deletions, insertions and substitutions in the polypeptidesdescribed herein are not expected to produce radical changes in thecharacteristics of the polypeptide (e.g., the activity of thepolypeptide). However, when it is difficult to predict the exact effectof the substitution, deletion or insertion in advance of doing so, oneof skill in the art will appreciate that the effect can be evaluated byroutine screening assays that can screen for the particular polypeptideactivities of interest (e.g., conferring increased resistance to anematode plant pest, reducing the growth of a nematode plant pest,reducing nematode cyst development).

In some embodiments, the compositions of the invention can compriseactive fragments of the polypeptide. As used herein, “fragment” means aportion of the reference polypeptide that retains the polypeptideactivity of conferring increased resistance to a nematode plant pest,reducing the growth of a nematode plant pest, reducing cyst development.A fragment also means a portion of a nucleic acid molecule encoding thereference polypeptide. An active fragment of the polypeptide can beprepared, for example, by isolating a portion of a polypeptide-encodingnucleic acid molecule that expresses the encoded fragment of thepolypeptide (e.g., by recombinant expression in vitro), and assessingthe activity of the fragment. Nucleic acid molecules encoding suchfragments can be at least about 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, or 2000 contiguous nucleotides, or up to thenumber of nucleotides present in a full-length polypeptide-encodingnucleic acid molecule. As such, polypeptide fragments can be at leastabout 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 525, 550, 600, 625,650, 675, or 700 contiguous amino acid residues, or up to the totalnumber of amino acid residues present in the full-length polypeptide.

Thus, in some embodiments, a variant or functional fragment of apolypeptide of this invention or a variant or functional fragment havingsubstantial identity to a polypeptide sequence of this invention (e.g.,SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ IDNOs:665-1046) when produced in a transgenic plant reduces the ability ofnematode pests to survive, grow and reproduce in/on or around thetransgenic plant, or reduces nematode-related damage or loss to thetransgenic plants producing said polypeptides.

In some embodiments, the nematicidal composition further comprises anagriculturally acceptable carrier. As used herein an“agriculturally-acceptable carrier” can include natural, synthetic,organic and/or inorganic material which is combined with the activecomponent to facilitate its application to the plant, or part thereof.An agriculturally-acceptable carrier includes, but is not limited to,inert components, dispersants, surfactants, adjuvants, tackifiers,stickers, binders, or combinations thereof, that can be used inagricultural formulations. In other embodiments, as agriculturallyacceptable carrier can be a transgenic plant or plant part.

In some embodiments, the nematicidal composition can further compriseone or more additional nematicidal and/or insecticidal compounds. Suchnematicidal compounds include, without limitation, chloropicrin, metamsodium, metam potassium, dazomet, iodomethane, dimethyl disulfide(DMDS), sulfryl fluoride, oxamyl and fosthiazate.

In other embodiments, the nematicidal composition can further comprisepolypeptides having insecticidal activity. Such insecticidalpolypeptides include, without limitation, crystal (Cry) endotoxins fromBacillus thuringiensis and vegetative insecticidal proteins (VIPs) fromBacillus sp.

The polypeptides and compositions thereof of the invention can beapplied to the surface of a plant or plant part, including but notlimited to, seed, leaves, flowers, stems, tubers, roots, and the like.In some embodiments, the polypeptides and compositions of the inventionare delivered orally to a nematode, wherein the nematode ingests one ormore parts of a plant to which a composition comprising the polypeptidesof the invention has been applied. Applying the compositions of theinvention to a plant can be done using any method known to those ofskill in the art for applying compounds, compositions, formulations andthe like to plant surfaces. Some non-limiting examples of applyinginclude spraying, dusting, sprinkling, scattering, misting, atomizing,broadcasting, soaking, soil injection, soil incorporation, drenching(e.g., root, soil treatment), dipping, pouring, coating, leaf or steminfiltration, side dressing or seed treatment, and the like, andcombinations thereof. These and other procedures for applying acompound(s), composition(s) or formulation(s) to a plant or part thereofare well-known to those of skill in the art. In some embodiments, thepolypeptides are delivered orally to a nematode in the form of atransgenic plant comprising one or more nucleotide sequences encodingone or more polypeptides of the invention.

As used herein, the terms “express,” “expresses,” “expressed” or“expression,” and the like, with respect to a nucleic acid moleculeand/or a nucleotide sequence (e.g., RNA or DNA) indicates that thenucleic acid molecule and/or a nucleotide sequence is transcribed and,optionally, translated. Thus, a nucleic acid molecule and/or anucleotide sequence may express a polypeptide of interest or afunctional untranslated RNA.

A “heterologous” nucleotide sequence is a nucleotide sequence notnaturally associated with a host cell into which it is introduced,including non-naturally occurring multiple copies of a naturallyoccurring nucleotide sequence.

A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptideor amino acid sequence refers to a naturally occurring or endogenousnucleic acid, nucleotide sequence, polypeptide or amino acid sequence.Thus, for example, a “wild type mRNA” is an mRNA that is naturallyoccurring in or endogenous to the organism. A “homologous” nucleic acidsequence is a nucleotide sequence naturally associated with a host cellinto which it is introduced.

Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,”“nucleotide sequence” and “polynucleotide” can be used interchangeablyand encompass both RNA and DNA, including cDNA, genomic DNA, mRNA,synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNAand DNA. The term polynucleotide, nucleotide sequence, or nucleic acidrefers to a chain of nucleotides without regard to length of the chain.The nucleic acid can be double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid can be a sense strand or an antisensestrand. The nucleic acid can be synthesized using oligonucleotideanalogs or derivatives (e.g., inosine or phosphorothioate nucleotides).Such oligonucleotides can be used, for example, to prepare nucleic acidsthat have altered base-pairing abilities or increased resistance tonucleases. The present invention further provides a nucleic acid that isthe complement (which can be either a full complement or a partialcomplement) of a nucleic acid, nucleotide sequence, or polynucleotide ofthis invention. Nucleic acid molecules and/or nucleotide sequencesprovided herein are presented herein in the 5′ to 3′ direction, fromleft to right and are represented using the standard code forrepresenting the nucleotide characters as set forth in the U.S. sequencerules, 37 CFR §§ 1.821-1.825 and the World Intellectual PropertyOrganization (WIPO) Standard ST.25.

The term “antisense nucleotide sequence” or “antisense oligonucleotide”as used herein, refers to a nucleotide sequence that is complementary toa specified DNA or RNA sequence. Antisense oligonucleotides and nucleicacids that express the same can be made in accordance with conventionaltechniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No.5,149,797 to Pederson et al. The antisense nucleotide sequence can becomplementary to the entire nucleotide sequence encoding the polypeptideor a portion thereof of at least 10, 20, 40, 50, 75, 100, 150, 200, 300,or 500 contiguous bases and will reduce the level of polypeptideproduction.

Those skilled in the art will appreciate that it is not necessary thatthe antisense nucleotide sequence be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to hybridize to its target and reduceproduction of the polypeptide or transcript. As is known in the art, ahigher degree of sequence similarity is generally required for shortantisense nucleotide sequences, whereas a greater degree of mismatchedbases will be tolerated by longer antisense nucleotide sequences.

For example, hybridization of such nucleotide sequences can be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 35-40% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at37° C.; conditions represented by a wash stringency of 40-45% formamidewith 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and/orconditions represented by a wash stringency of 50% formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively) tothe nucleotide sequences specifically disclosed herein. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (ColdSpring Harbor, N.Y., 1989).

In other embodiments, antisense nucleotide sequences of the inventionhave at least about 70%, 80%, 90%, 95%, 97%, 98% or higher sequencesimilarity with the complement of the coding sequences specificallydisclosed herein and will reduce the level of polypeptide production.

In other embodiments, the antisense nucleotide sequence can be directedagainst any coding sequence, the silencing of which results in amodulation of a polypeptide of this invention (e.g., SEQ ID NOs:29-42,135-209, 243-260, and/or 665-1046).

The length of the antisense nucleotide sequence (i.e., the number ofnucleotides therein) is not critical as long as it binds selectively tothe intended location and reduces transcription and/or translation ofthe target sequence, and can be determined in accordance with routineprocedures. In general, the antisense nucleotide sequence will be fromabout eight, ten or twelve nucleotides in length to about 20, 30, 50,75, 100, 200, 300, 400 nucleotides, or longer, in length.

An antisense nucleotide sequence can be constructed using chemicalsynthesis and enzymatic ligation reactions by procedures known in theart. For example, an antisense nucleotide sequence can be chemicallysynthesized using naturally occurring nucleotides or various modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleotide sequences, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleotide sequence include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomet-hyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleotide sequencecan be produced using an expression vector into which a nucleic acid hasbeen cloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest).

The antisense nucleotide sequences of the invention further includenucleotide sequences wherein at least one, or all, of theinternucleotide bridging phosphate residues are modified phosphates,such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues can be modified as described. In another non-limiting example,the antisense nucleotide sequence is a nucleotide sequence in which one,or all, of the nucleotides contain a 2′ lower alkyl moiety (e.g., Ci-C4,linear or branched, saturated or unsaturated alkyl, such as methyl,ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). Forexample, every other one of the nucleotides can be modified asdescribed. See also, Furdon et al., Nucleic Acids Res. 17:9193 (1989);Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al.,Nucleic Acids Res. 18:3537 (1990); Sproat et al., Nucleic Acids Res.17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011(1988); incorporated by reference herein for their teaching of methodsof making antisense molecules, including those containing modifiednucleotide bases).

Triple helix base-pairing methods can also be employed to inhibitproduction of polypeptides of this invention (e.g., SEQ ID NOs:29-42,135-209, 243-260, and/or 665-1046). Triple helix pairing is believed towork by inhibiting the ability of the double helix to open sufficientlyfor the binding of polymerases, transcription factors, or regulatorymolecules. Recent therapeutic advances using triplex DNA have beendescribed in the literature (e.g., Gee et al., (1994) In: Huber et al.,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.).

Different nucleic acids or proteins having homology are referred toherein as “homologues.” The term homologue includes homologous sequencesfrom the same and other species and orthologous sequences from the sameand other species. “Homology” refers to the level of similarity betweentwo or more nucleic acid and/or amino acid sequences in terms of percentof positional identity (i.e., sequence similarity or identity). Homologyalso refers to the concept of similar functional properties amongdifferent nucleic acids or proteins. Thus, the compositions and methodsof the invention further comprise homologues to the nucleotide sequencesand polypeptide sequences of this invention. “Orthologous,” as usedherein, refers to homologous nucleotide sequences and/or amino acidsequences in different species that arose from a common ancestral geneduring speciation. A homologue of this invention has a significantsequence identity (e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or100%) to the nucleotide sequences of the invention.

A homologue as described herein can be utilized with any composition ormethod of the invention, alone or in combination with one another and/orwith one or more nucleotide sequences or polypeptide sequences of theinvention. Thus, in one embodiment, the invention provides a nucleicacid molecule comprising, consisting essentially of, or consisting ofone or more nucleotide sequences of the invention and/or one or morehomologues of any nucleotide sequence of the invention. In a furtherembodiment, the invention provides polypeptide compositions comprising,consisting essentially of, or consisting of one or more of thepolypeptides of this invention and/or a homologue thereof.

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or peptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. “Identity” can be readily calculated by known methodsincluding, but not limited to, those described in: ComputationalMolecular Biology (Lesk, A. M., ed.) Oxford University Press, New York(1988); Biocomputing: Informatics and Genome Projects (Smith, D. W.,ed.) Academic Press, New York (1993); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press,New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje,G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov,M. and Devereux, J., eds.) Stockton Press, New York (1991).

As used herein, the term “percent sequence identity” or “percentidentity” refers to the percentage of identical nucleotides in a linearpolynucleotide sequence of a reference (“query”) polynucleotide molecule(or its complementary strand) as compared to a test (“subject”)polynucleotide molecule (or its complementary strand) when the twosequences are optimally aligned. In some embodiments, “percent identity”can refer to the percentage of identical amino acids in an amino acidsequence.

As used herein, the phrase “substantially identical,” in the context oftwo nucleic acid molecules, nucleotide sequences or protein sequences,refers to two or more sequences or subsequences that have at least about70%, at least about 75%, at least about 80%, at least about 81%, atleast about 82%, at least about 83%, at least about 84%, at least about85%, at least about 86%, at least about 87%, at least about 88%, atleast about 89%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. In some embodiments ofthe invention, the substantial identity exists over a region of thesequences that is at least about 50 residues to about 150 residues inlength. Thus, in some embodiments of the invention, the substantialidentity exists over a region of the sequences that is at least about50, about 60, about 70, about 80, about 90, about 100, about 110, about120, about 130, about 140, about 150, or more residues in length. Insome particular embodiments, the sequences are substantially identicalover at least about 150 residues. In a further embodiment, the sequencesare substantially identical over the entire length of the codingregions. Furthermore, in representative embodiments, substantiallyidentical nucleotide or protein sequences perform substantially the samefunction (e.g., conferring increased resistance to a nematode plantpest, reducing the growth of a nematode plant pest, reducing nematodecyst development).

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for aligning a comparison window are wellknown to those skilled in the art and may be conducted by tools such asthe local homology algorithm of Smith and Waterman, the homologyalignment algorithm of Needleman and Wunsch, the search for similaritymethod of Pearson and Lipman, and optionally by computerizedimplementations of these algorithms such as GAP, BESTFIT, FASTA, andTFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc.,San Diego, Calif.). An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents which are shared by the two aligned sequences divided by thetotal number of components in the reference sequence segment, i.e., theentire reference sequence or a smaller defined part of the referencesequence. Percent sequence identity is represented as the identityfraction multiplied by 100. The comparison of one or more polynucleotidesequences may be to a full-length polynucleotide sequence or a portionthereof, or to a longer polynucleotide sequence. For purposes of thisinvention “percent identity” may also be determined using BLASTX version2.0 for translated nucleotide sequences and BLASTN version 2.0 forpolynucleotide sequences.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleotide sequenceto the reference nucleotide sequence is less than about 0.1 to less thanabout 0.001. Thus, in some embodiments of the invention, the smallestsum probability in a comparison of the test nucleotide sequence to thereference nucleotide sequence is less than about 0.001.

Two nucleotide sequences can also be considered to be substantiallyidentical when the two sequences hybridize to each other under stringentconditions. In some representative embodiments, two nucleotide sequencesconsidered to be substantially identical hybridize to each other underhighly stringent conditions.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. An extensiveguide to the hybridization of nucleic acids is found in TijssenLaboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” Elsevier, New York (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleotide sequences which have morethan 100 complementary residues on a filter in a Southern or northernblot is 50% formamide with 1 mg of heparin at 42° C., with thehybridization being carried out overnight. An example of highlystringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes.An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for15 minutes (see, Sambrook, infra, for a description of SSC buffer).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of a medium stringency washfor a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for15 minutes. An example of a low stringency wash for a duplex of, e.g.,more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. Forshort probes (e.g., about 10 to 50 nucleotides), stringent conditionstypically involve salt concentrations of less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleotide sequences that do not hybridize to each otherunder stringent conditions are still substantially identical if theproteins that they encode are substantially identical. This can occur,for example, when a copy of a nucleotide sequence is created using themaximum codon degeneracy permitted by the genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone homologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of theinvention. In one embodiment, a reference nucleotide sequence hybridizesto the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50°C. In another embodiment, the reference nucleotide sequence hybridizesto the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50°C. or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C. In still furtherembodiments, the reference nucleotide sequence hybridizes to the “test”nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., or in 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 0.1×SSC, 0.1% SDS at 65° C.

In particular embodiments, a further indication that two nucleotidesequences or two polypeptide sequences are substantially identical canbe that the protein encoded by the first nucleic acid is immunologicallycross reactive with, or specifically binds to, the protein encoded bythe second nucleic acid. Thus, in some embodiments, a polypeptide can besubstantially identical to a second polypeptide, for example, where thetwo polypeptides differ only by conservative substitutions.

In some embodiments, the recombinant nucleic acids molecules, nucleotidesequences and polypeptides of the invention are “isolated.” An“isolated” nucleic acid molecule, an “isolated” nucleotide sequence oran “isolated” polypeptide is a nucleic acid molecule, nucleotidesequence or polypeptide that, by the hand of man, exists apart from itsnative environment and is therefore not a product of nature. An isolatednucleic acid molecule, nucleotide sequence or polypeptide may exist in apurified form that is at least partially separated from at least some ofthe other components of the naturally occurring organism or virus, forexample, the cell or viral structural components or other polypeptidesor nucleic acids commonly found associated with the polynucleotide. Inrepresentative embodiments, the isolated nucleic acid molecule, theisolated nucleotide sequence and/or the isolated polypeptide is at leastabout 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or morepure.

In other embodiments, an isolated nucleic acid molecule, nucleotidesequence or polypeptide may exist in a non-native environment such as,for example, a recombinant host cell. Thus, for example, with respect tonucleotide sequences, the term “isolated” means that it is separatedfrom the chromosome and/or cell in which it naturally occurs. Apolynucleotide is also isolated if it is separated from the chromosomeand/or cell in which it naturally occurs in and is then inserted into agenetic context, a chromosome and/or a cell in which it does notnaturally occur (e.g., a different host cell, different regulatorysequences, and/or different position in the genome than as found innature). Accordingly, the recombinant nucleic acid molecules, nucleotidesequences and their encoded polypeptides are “isolated” in that, by thehand of man, they exist apart from their native environment andtherefore are not products of nature, however, in some embodiments, theycan be introduced into and exist in a recombinant host cell.

In some embodiments, the nucleotide sequences and/or nucleic acidmolecules of the invention can be operatively associated with a varietyof promoters for expression in host cells (e.g., plant cells). Thus, insome embodiments, the invention provides transformed host cells andtransformed organisms comprising the transformed host cells, wherein thehost cells and organisms are transformed with one or more nucleic acidmolecules/nucleotide sequences of the invention. As used herein,“operatively associated with,” when referring to a first nucleic acidsequence that is operatively linked to a second nucleic acid sequence,means a situation when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operatively associated with a coding sequence ifthe promoter effects the transcription or expression of the codingsequence.

A DNA “promoter” is an untranslated DNA sequence upstream of a codingregion that contains the binding site for RNA polymerase and initiatestranscription of the DNA. A “promoter region” can also include otherelements that act as regulators of gene expression. Promoters caninclude, for example, constitutive, inducible, temporally regulated,developmentally regulated, chemically regulated, tissue-preferred andtissue-specific promoters for use in the preparation of recombinantnucleic acid molecules, i.e., chimeric genes. In particular aspects, a“promoter” useful with the invention is a promoter capable of initiatingtranscription of a nucleotide sequence in a cell of a plant.

A “chimeric gene” is a recombinant nucleic acid molecule in which apromoter or other regulatory nucleotide sequence is operativelyassociated with a nucleotide sequence that codes for an mRNA or which isexpressed as a protein, such that the regulatory nucleotide sequence isable to regulate transcription or expression of the associatednucleotide sequence. The regulatory nucleotide sequence of the chimericgene is not normally operatively linked to the associated nucleotidesequence as found in nature.

The choice of promoter will vary depending on the temporal and spatialrequirements for expression, and also depending on the host cell to betransformed. Thus, for example, expression of the nucleotide sequencesof the invention can be in any plant and/or plant part, (e.g., inleaves, in stalks or stems, in ears, in inflorescences (e.g. spikes,panicles, cobs, etc.), in roots, seeds and/or seedlings, and the like).In many cases, however, protection against more than one type of pest issought, and thus expression in multiple tissues is desirable. Althoughmany promoters from dicotyledons have been shown to be operational inmonocotyledons and vice versa, ideally dicotyledonous promoters areselected for expression in dicotyledons, and monocotyledonous promotersfor expression in monocotyledons. However, there is no restriction tothe provenance of selected promoters; it is sufficient that they areoperational in driving the expression of the nucleotide sequences in thedesired cell.

Promoters useful with the invention include, but are not limited to,those that drive expression of a nucleotide sequence constitutively,those that drive expression when induced, and those that driveexpression in a tissue- or developmentally-specific manner. Thesevarious types of promoters are known in the art.

Examples of constitutive promoters include, but are not limited to,cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well asU.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol.9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci.USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990)Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter.The constitutive promoter derived from ubiquitin accumulates in manycell types. Ubiquitin promoters have been cloned from several plantspecies for use in transgenic plants, for example, sunflower (Binet etal., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989.Plant Molec. Biol. 12: 619-632), and arabidopsis (Norris et al. 1993.Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter (UbiP) hasbeen developed in transgenic monocot systems and its sequence andvectors constructed for monocot transformation are disclosed in thepatent publication EP 0 342 926. The ubiquitin promoter is suitable forthe expression of the nucleotide sequences of the invention intransgenic plants, especially monocotyledons. Further, the promoterexpression cassettes described by McElroy et al. (Mol. Gen. Genet. 231:150-160 (1991)) can be easily modified for the expression of thenucleotide sequences of the invention and are particularly suitable foruse in monocotyledonous hosts.

In some embodiments, tissue specific/tissue preferred promoters can beused. Tissue specific or preferred expression patterns include, but arenot limited to, green tissue specific or preferred, root specific orpreferred, stem specific or preferred, and flower specific or preferred.Promoters suitable for expression in green tissue include many thatregulate genes involved in photosynthesis and many of these have beencloned from both monocotyledons and dicotyledons. In one embodiment, apromoter useful with the invention is the maize PEPC promoter from thephosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol.12:579-589 (1989)). Non-limiting examples of tissue-specific promotersinclude those associated with genes encoding the seed storage proteins(such as β-conglycinin, cruciferin, napin and phaseolin), zein or oilbody proteins (such as oleosin), or proteins involved in fatty acidbiosynthesis (including acyl carrier protein, stearoyl-ACP desaturaseand fatty acid desaturases (fad 2-1)), and other nucleic acids expressedduring embryo development (such as Bce4, see, e.g., Kridl et al. (1991)Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378).Tissue-specific or tissue-preferential promoters useful for theexpression of the nucleotide sequences of the invention in plants,particularly maize, include but are not limited to those that directexpression in root, pith, leaf or pollen. Such promoters are disclosed,for example, in WO 93/07278, herein incorporated by reference in itsentirety. Other non-limiting examples of tissue specific or tissuepreferred promoters useful with the invention the cotton rubiscopromoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthasepromoter disclosed in U.S. Pat. No. 5,604,121; the root specificpromoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452 269to Ciba-Geigy); the stem specific promoter described in U.S. Pat. No.5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpAgene; and the cestrum yellow leaf curling virus promoter disclosed in WO01/73087, all incorporated by reference

Additional examples of tissue-specific/tissue preferred promotersinclude, but are not limited to, the root-specific promoters RCc3 (Jeonget al. Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Pat. No.5,459,252), the lectin promoter (Lindstrom et al. (1990) Der. Genet.11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), cornalcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic AcidsRes. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (VanderMijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115),corn light harvesting complex promoter (Bansal et al. (1992) Proc. Natl.Acad. Sci. USA 89:3654-3658), corn heat shock protein promoter (O'Dellet al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J.5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore,“Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphatecarboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaendered., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet.205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al.(1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopalinesynthase promoter (Langridge et al. (1989), supra), petunia chalconeisomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), beanglycine rich protein 1 promoter (Keller et al. (1989) Genes Dev.3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol.Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) NucleicAcids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen.Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina etal. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic AcidsRes. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354),globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872),α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet.215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol.12:579-589), R gene complex-associated promoters (Chandler et al. (1989)Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al.(1991) EMBO J. 10:2605-2612). In some particular embodiments, thenucleotide sequences of the invention are operatively associated with aroot-preferred promoter.

Particularly useful for seed-specific expression is the pea vicilinpromoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as theseed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Usefulpromoters for expression in mature leaves are those that are switched onat the onset of senescence, such as the SAG promoter from Arabidopsis(Gan et al. (1995) Science 270:1986-1988).

In addition, promoters functional in plastids can be used. Non-limitingexamples of such promoters include the bacteriophage T3 gene 9 5′ UTRand other promoters disclosed in U.S. Pat. No. 7,579,516. Otherpromoters useful with the invention include but are not limited to theS-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsininhibitor gene promoter (Kti3).

In some embodiments of the invention, inducible promoters can be used.Thus, for example, chemical-regulated promoters can be used to modulatethe expression of a gene in a plant through the application of anexogenous chemical regulator. Regulation of the expression of nucleotidesequences of the invention via promoters that are chemically regulatedenables the polypeptides of the invention to be synthesized only whenthe crop plants are treated with the inducing chemicals. Depending uponthe objective, the promoter may be a chemical-inducible promoter, whereapplication of a chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression.

Chemical inducible promoters are known in the art and include, but arenot limited to, the maize In2-2 promoter, which is activated bybenzenesulfonamide herbicide safeners, the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides, and the tobacco PR-1a promoter, which isactivated by salicylic acid (e.g., the PR1a system), steroidsteroid-responsive promoters (see, e.g., the glucocorticoid-induciblepromoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88,10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see,e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, Lac repressor system promoters,copper-inducible system promoters, salicylate-inducible system promoters(e.g., the PR1a system), glucocorticoid-inducible promoters (Aoyama etal. (1997) Plant J. 11:605-612), and ecdysone-inducible systempromoters.

Other non-limiting examples of inducible promoters include ABA- andturgor-inducible promoters, the auxin-binding protein gene promoter(Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoidglycosyl-transferase promoter (Ralston et al. (1988) Genetics119:185-197), the MPI proteinase inhibitor promoter (Cordero et al.(1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphatedehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol.29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; andQuigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are thebenzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) andalcohol-inducible (Int'l Patent Application Publication Nos. WO 97/06269and WO 97/06268) systems and glutathione 5-transferase promoters.Likewise, one can use any of the inducible promoters described in Gatz(1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev.Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically induciblepromoters useful for directing the expression of the nucleotidesequences of this invention in plants are disclosed in U.S. Pat. No.5,614,395 herein incorporated by reference in its entirety. Chemicalinduction of gene expression is also detailed in the publishedapplication EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. Insome embodiments, a promoter for chemical induction can be the tobaccoPR-1a promoter.

In further aspects, the nucleotide sequences of the invention can beoperatively associated with a promoter that is wound inducible orinducible by pest or pathogen infection (e.g., a nematode plant pest).Numerous promoters have been described which are expressed at woundsites and/or at the sites of pest attack (e.g., insect/nematode feeding)or phytopathogen infection. Ideally, such a promoter should be activeonly locally at or adjacent to the sites of attack, and in this wayexpression of the nucleotide sequences of the invention will be focusedin the cells that are being invaded. Such promoters include, but are notlimited to, those described by Stanford et al., Mol. Gen. Genet.215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993),Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, PlantMolec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol.22:129-142 (1993), Warner et al. Plant J. 3:191-201 (1993), U.S. Pat.Nos. 5,750,386, 5,955,646, 6,262,344, 6,395,963, 6,703,541, 7,078,589,7,196,247, 7,223,901, and U.S. Patent Application Publication2010043102.

As used herein, “expression cassette” means a nucleic acid moleculecomprising a nucleotide sequence of interest (e.g., the nucleotidesequences of the invention), wherein said nucleotide sequence isoperatively associated with at least a control sequence (e.g., apromoter). Thus, some embodiments of the invention provide expressioncassettes designed to express the nucleotides sequences of theinvention. In this manner, for example, one or more plant promotersoperatively associated with one or more nucleotide sequences of theinvention (e.g., SEQ ID NOs:1-28, SEQ ID NOs:43-134, SEQ ID NOs:210-242,SEQ ID NOs:261-644) are provided in expression cassettes for expressionin an organism or cell thereof (e.g., a plant, plant part and/or plantcell).

An expression cassette comprising a nucleotide sequence of interest maybe chimeric, meaning that at least one of its components is heterologouswith respect to at least one of its other components. An expressioncassette may also be one that is naturally occurring but has beenobtained in a recombinant form useful for heterologous expression.Typically, however, the expression cassette is heterologous with respectto the host, i.e., the particular nucleic acid sequence of theexpression cassette does not occur naturally in the host cell and musthave been introduced into the host cell or an ancestor of the host cellby a transformation event.

In addition to the promoters operatively linked to the nucleotidesequences of the invention, an expression cassette of the invention canalso include other regulatory sequences. As used herein, “regulatorysequences” means nucleotide sequences located upstream (5′ non-codingsequences), within or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences include, but are not limited to, promoters, enhancers,introns, translation leader sequences, termination signals, andpolyadenylation signal sequences.

For purposes of the invention, the regulatory sequences or regions canbe native/analogous to the plant, plant part and/or plant cell and/orthe regulatory sequences can be native/analogous to the other regulatorysequences. Alternatively, the regulatory sequences may be heterologousto the plant (and/or plant part and/or plant cell) and/or to each other(i.e., the regulatory sequences). Thus, for example, a promoter can beheterologous when it is operatively linked to a polynucleotide from aspecies different from the species from which the polynucleotide wasderived. Alternatively, a promoter can also be heterologous to aselected nucleotide sequence if the promoter is from the same/analogousspecies from which the polynucleotide is derived, but one or both (i.e.,promoter and/or polynucleotide) are substantially modified from theiroriginal form and/or genomic locus, and/or the promoter is not thenative promoter for the operably linked polynucleotide.

A number of non-translated leader sequences derived from viruses areknown to enhance gene expression. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “ω-sequence”), Maize Chlorotic MottleVirus (MCMV) and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (Gallie et al. (1987) Nucleic AcidsRes. 15:8693-8711; and Skuzeski et al. (1990) Plant Mol. Biol.15:65-79). Other leader sequences known in the art include, but are notlimited to, picornavirus leaders such as an encephalomyocarditis (EMCV)5′ noncoding region leader (Elroy-Stein et al. (1989) Proc. Natl. Acad.Sci. USA 86:6126-6130); potyvirus leaders such as a Tobacco Etch Virus(TEV) leader (Allison et al. (1986) Virology 154:9-20); Maize DwarfMosaic Virus (MDMV) leader (Allison et al. (1986), supra); humanimmunoglobulin heavy-chain binding protein (BiP) leader (Macejak & Samow(1991) Nature 353:90-94); untranslated leader from the coat protein mRNAof AMV (AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622-625); tobaccomosaic TMV leader (Gallie et al. (1989) Molecular Biology of RNA237-256); and MCMV leader (Lommel et al. (1991) Virology 81:382-385).See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

An expression cassette also can optionally include a transcriptionaland/or translational termination region (i.e., termination region) thatis functional in plants. A variety of transcriptional terminators areavailable for use in expression cassettes and are responsible for thetermination of transcription beyond the heterologous nucleotide sequenceof interest and correct mRNA polyadenylation. The termination region maybe native to the transcriptional initiation region, may be native to theoperably linked nucleotide sequence of interest, may be native to theplant host, or may be derived from another source (i.e., foreign orheterologous to the promoter, the nucleotide sequence of interest, theplant host, or any combination thereof). Appropriate transcriptionalterminators include, but are not limited to, the CAMV 35S terminator,the tml terminator, the nopaline synthase terminator and/or the pea rbcsE9 terminator. These can be used in both monocotyledons anddicotyledons. In addition, a coding sequence's native transcriptionterminator can be used.

An expression cassette of the invention also can include a nucleotidesequence for a selectable marker, which can be used to select atransformed plant, plant part and/or plant cell. As used herein,“selectable marker” means a nucleotide sequence that when expressedimparts a distinct phenotype to the plant, plant part and/or plant cellexpressing the marker and thus allows such transformed plants, plantparts and/or plant cells to be distinguished from those that do not havethe marker. Such a nucleotide sequence may encode either a selectable orscreenable marker, depending on whether the marker confers a trait thatcan be selected for by chemical means, such as by using a selectiveagent (e.g., an antibiotic, herbicide, or the like), or on whether themarker is simply a trait that one can identify through observation ortesting, such as by screening (e.g., the R-locus trait). Of course, manyexamples of suitable selectable markers are known in the art and can beused in the expression cassettes described herein.

Examples of selectable markers include, but are not limited to, anucleotide sequence encoding neo or nptII, which confers resistance tokanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet.199:183-188); a nucleotide sequence encoding bar, which confersresistance to phosphinothricin; a nucleotide sequence encoding analtered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, whichconfers resistance to glyphosate (Hinchee et al. (1988) Biotech.6:915-922); a nucleotide sequence encoding a nitrilase such as bxn fromKlebsiella ozaenae that confers resistance to bromoxynil (Stalker et al.(1988) Science 242:419-423); a nucleotide sequence encoding an alteredacetolactate synthase (ALS) that confers resistance to imidazolinone,sulfonylurea or other ALS-inhibiting chemicals (EP Patent ApplicationNo. 154204); a nucleotide sequence encoding a methotrexate-resistantdihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem.263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenasethat confers resistance to dalapon; a nucleotide sequence encoding amannose-6-phosphate isomerase (also referred to as phosphomannoseisomerase (PMI)) that confers an ability to metabolize mannose (U.S.Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding analtered anthranilate synthase that confers resistance to 5-methyltryptophan; and/or a nucleotide sequence encoding hph that confersresistance to hygromycin. One of skill in the art is capable of choosinga suitable selectable marker for use in an expression cassette of theinvention.

Additional selectable markers include, but are not limited to, anucleotide sequence encoding β-glucuronidase or uidA (GUS) that encodesan enzyme for which various chromogenic substrates are known; an R-locusnucleotide sequence that encodes a product that regulates the productionof anthocyanin pigments (red color) in plant tissues (Dellaporta et al.,“Molecular cloning of the maize R-nj allele by transposon-tagging withAc,” pp. 263-282 In: Chromosome Structure and Function: Impact of NewConcepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds.,Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, anenzyme for which various chromogenic substrates are known (e.g., PADAC,a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci.USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes acatechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone, which in turncondenses to form melanin (Katz et al. (1983) J. Gen. Microbiol.129:2703-2714); a nucleotide sequence encoding β-galactosidase, anenzyme for which there are chromogenic substrates; a nucleotide sequenceencoding luciferase (lux) that allows for bioluminescence detection (Owet al. (1986) Science 234:856-859); a nucleotide sequence encodingaequorin, which may be employed in calcium-sensitive bioluminescencedetection (Prasher et al. (1985) Biochem. Biophys. Res. Comm.126:1259-1268); or a nucleotide sequence encoding green fluorescentprotein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One ofskill in the art is capable of choosing a suitable selectable marker foruse in an expression cassette of the invention.

An expression cassette of the invention also can include nucleotidesequences that encode other desired traits. Such desired traits can beother nucleotide sequences which confer nematode resistance, insectresistance, or which confer other agriculturally desirable traits. Suchnucleotide sequences can be stacked with any combination of nucleotidesequences to create plants, plant parts or plant cells having thedesired phenotype. Stacked combinations can be created by any methodincluding, but not limited to, cross breeding plants by any conventionalmethodology, or by genetic transformation. If stacked by geneticallytransforming the plants, nucleotide sequences encoding additionaldesired traits can be combined at any time and in any order. Forexample, a transgenic plant comprising one or more desired traits can beused as the target to introduce further traits by subsequenttransformation. The additional nucleotide sequences can be introducedsimultaneously in a co-transformation protocol with a nucleotidesequence, nucleic acid molecule, nucleic acid construct, and/orcomposition of the invention, provided by any combination of expressioncassettes. For example, if two nucleotide sequences will be introduced,they can be incorporated in separate cassettes (trans) or can beincorporated on the same cassette (cis). Expression of the nucleotidesequences can be driven by the same promoter or by different promoters.It is further recognized that nucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, e.g., Int'l Patent Application Publication Nos. WO 99/25821; WO99/25854; WO 99/25840; WO 99/25855 and WO 99/25853.

Thus, an expression cassette can include a coding sequence for one ormore polypeptides for agronomic traits that primarily are of benefit toa seed company, grower or grain processor. A polypeptide of interest canbe any polypeptide encoded by a nucleotide sequence of interest.Non-limiting examples of polypeptides of interest that are suitable forproduction in plants include those resulting in agronomically importanttraits such as herbicide resistance (also sometimes referred to as“herbicide tolerance”), virus resistance, bacterial pathogen resistance,insect resistance, nematode resistance, and/or fungal resistance. See,e.g., U.S. Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and6,337,431. Thus, in some embodiments, the expression cassette orexpression vector of the invention can comprise one or more nucleotidesequences that confer insect resistance and/or additional nematoderesistance.

In other embodiments, a polypeptide of interest also can be one thatincreases plant vigor or yield (including traits that allow a plant togrow at different temperatures, soil conditions and levels of sunlightand precipitation), or one that allows identification of a plantexhibiting a trait of interest (e.g., a selectable marker, seed coatcolor, etc.). Various polypeptides of interest, as well as methods forintroducing these polypeptides into a plant, are described, for example,in U.S. Pat. Nos. 4,761,373; 4,769,061; 4,810,648; 4,940,835; 4,975,374;5,013,659; 5,162,602; 5,276,268; 5,304,730; 5,495,071; 5,554,798;5,561,236; 5,569,823; 5,767,366; 5,879,903, 5,928,937; 6,084,155;6,329,504 and 6,337,431; as well as US Patent Publication No.2001/0016956. See also, on the World Wide Web atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.

In some particular embodiments of the invention, a nucleotide sequenceof interest includes, but is not limited to, RNAi (siRNA, antisense RNA)and/or miRNA known to be associated with nematode resistance, and/ornucleotide sequences coding for insect resistance including, but notlimited to, nucleotide sequences coding for Bacillus thuringiensis (Bt)toxins, for example, the various delta-endotoxin genes such as Cry1Aa,Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9Cand Cry9B; as well as genes encoding vegetative insecticidal proteinssuch as Vip1, Vip2 and Vip3). An extensive list of Bt toxins can befound on the worldwide web at Bacillus thuringiensis Toxin NomenclatureDatabase maintained by the University of Sussex (see also, Crickmore etal. (1998) Microbiol. Mol. Biol. Rev. 62:807-813).

In addition to expression cassettes, the nucleic acid molecules andnucleotide sequences described herein can be used in connection withvectors. The term “vector” refers to a composition for transferring,delivering or introducing a nucleic acid (or nucleic acids) into a cell.A vector comprises a nucleic acid molecule comprising the nucleotidesequence(s) to be transferred, delivered or introduced. Vectors for usein transformation of plants and other organisms are well known in theart. Non-limiting examples of general classes of vectors include a viralvector including but not limited to an adenovirus vector, a retroviralvector, an adeno-associated viral vector, a plasmid vector, a phagevector, a phagemid vector, a cosmid, a fosmid, a bacteriophage, or anartificial chromosome. The selection of a vector will depend upon thepreferred transformation technique and the target species fortransformation. Accordingly, in further embodiments, a recombinantnucleic acid molecule of the invention can be comprised within arecombinant vector. The size of a vector can vary considerably dependingon whether the vector comprises one or multiple expression cassettes(e.g., for molecular stacking). Thus, a vector size can range from about3 kb to about 30 kb. Thus, in some embodiments, a vector is about 3 kb,4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb,15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, or any range therein, in size. Insome particular embodiments, a vector can be about 3 kb to about 10 kbin size.

In additional embodiments of the invention, a method of producing atransgenic plant cell is provided, said method comprising introducinginto a plant cell a recombinant nucleic acid molecule/nucleotidesequence of the invention, thereby producing a transgenic plant cellthat can regenerate a transgenic plant having increased resistance to anematode plant pest as compared to a plant regenerated from a plant cellthat does not comprise said nucleic acid molecule. In some embodiments,the transgenic plant cell comprises more than one (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, etc.) nucleic acid molecule/nucleotide sequence of theinvention. Thus, in some aspects of the invention, the transgenicplants, or parts thereof, comprise and express one or more nucleic acidmolecule/nucleotide sequences of the invention, thereby producing one ormore polypeptides of the invention.

In representative embodiments, a method of producing a transgenic plantcell is provided, said method comprising introducing into a plant cell arecombinant nucleic acid molecule of the invention, said recombinantnucleic acid molecule comprising a nucleotide sequence operativelylinked to a promoter, which when expressed in a plant confer increasedresistance to a nematode plant pest, the nucleotide sequence comprising,consisting essentially of, or consisting of: (a) a nucleotide sequenceof SEQ ID NOs:1-28, SEQ ID NOs:43-134, SEQ ID NOs:210-242, SEQ IDNOs:261-644; (b) a nucleotide sequence that encodes a polypeptidecomprising, consisting essentially of, or consisting of the amino acidsequence of any one of SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ IDNOs:243-260, SEQ ID NOs:665-1046; (c) a nucleotide sequence havingsignificant sequence identity to nucleotide sequence of (a) and (b)above; (d) a nucleotide sequence which anneals under stringenthybridization conditions to the nucleotide sequence of (a), (b) or (c);(e) a nucleotide sequence that differs from the nucleotide sequences of(a), (b), (c) or (d) above due to the degeneracy of the genetic code; or(f) any combination of the nucleotide sequences of (a)-(e), therebyproducing a transgenic plant cell that can regenerate a plant havingincreased resistance to a nematode plant pest as compared to a plantregenerated from a plant cell that does not comprise said recombinantnucleic acid molecule. In further embodiments, a method of producing atransgenic plant cell is provided, said method comprising introducinginto a plant cell a recombinant nucleic acid molecule of the invention,said recombinant nucleic acid molecule comprising a nucleotide sequenceoperatively linked to a promoter, which when expressed in a plant conferincreased resistance to a nematode plant pest, the nucleotide sequencecomprising, consisting essentially of, or consisting of: (a) anucleotide sequence of SEQ ID NOs: 15, 17, 20, 22, 23, 24, 26, 226, 227,228, 230, 232 and/or 233; (b) a nucleotide sequence that encodes apolypeptide comprising, consisting essentially of, or consisting of theamino acid sequence of any one of SEQ ID NOs: 29, 31, 34, 36-38, 40,244-246, 250, 251; (c) a nucleotide sequence having significant sequenceidentity to nucleotide sequence of (a) and (b) above; (d) a nucleotidesequence which anneals under stringent hybridization conditions to thenucleotide sequence of (a), (b) or (c); (e) a nucleotide sequence thatdiffers from the nucleotide sequences of (a), (b), (c) or (d) above dueto the degeneracy of the genetic code; or (f) any combination of thenucleotide sequences of (a)-(e), thereby producing a transgenic plantcell that can regenerate a plant having increased resistance to anematode plant pest as compared to a plant regenerated from a plant cellthat does not comprise said recombinant nucleic acid molecule.

Thus, in some embodiments, the invention provides a transgenic plant orpart thereof that is regenerated from the transgenic plant cell of theinvention, wherein the transgenic plant or plant part has increasedresistance to a nematode plant pest as compared to a control plant orplant part that is regenerated from a plant cell that does not comprisesaid recombinant nucleic acid molecule.

The terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,”“enhancing,” and “enhancement” (and grammatical variations thereof), asused herein, describe an increase in the resistance of a plant to anematode plant pest (e.g., a soybean plant having increased resistanceto the soybean cyst nematode) by the introduction of a recombinantnucleic acid molecule of the invention into the plant, thereby producinga transgenic plant having increased resistance to the pest. Thisincrease in resistance can be observed by comparing the resistance ofthe plant transformed with the recombinant nucleic acid molecule of theinvention to the resistance of a plant lacking (i.e., not transformedwith) the recombinant nucleic acid molecule of the invention (i.e., acontrol).

As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,”“diminish,” “suppress,” and “decrease” (and grammatical variationsthereof), describe, for example, a decrease in the growth of a nematodeplant pest, a decrease in the ability of the nematode to survive, grow,feed, and/or reproduce, a decrease in the infectivity of a nematodeplant pest, a decrease in the infestation of a plant by a nematode plantpest, and/or a decrease in nematode cyst development by a nematode plantpest on roots of a plant as compared to a control as described herein.

A further aspect of the invention provides transformed non-human hostcells and transformed non-human organisms comprising the transformednon-human cells, wherein the transformed cells and transformed organismscomprise nucleic acid molecules comprising one or more nucleotidesequences of the invention. In some embodiments, the transformednon-human host cell includes but is not limited to a transformedbacterial cell, and/or a transformed plant cell. Thus, in someembodiments, the transformed non-human organism comprising thetransformed non-human host cell includes, but is not limited to, atransformed bacterium, and/or a transformed plant.

In some particular embodiments, the invention provides a transgenicplant cell comprising a nucleic acid molecule of the invention and/or atransgenic plant regenerated from said transgenic plant cell.Accordingly, in some embodiments of the invention, a transgenic planthaving increased resistance to a nematode plant pest is provided, saidtransgenic plant regenerated from a transgenic plant cell comprising atleast one recombinant nucleic acid molecule/nucleotide sequence of theinvention.

Additional aspects of the invention include a harvested product producedfrom the transgenic plants and/or parts thereof of the invention, aswell as a processed product produced from said harvested product. Aharvested product can be a whole plant or any plant part, as describedherein, wherein said harvested product comprises a recombinant nucleicacid molecule/nucleotide sequence of the invention. Thus, in someembodiments, non-limiting examples of a harvested product include aseed, a fruit, a flower or part thereof (e.g., an anther, a stigma, andthe like), a leaf, a stem, and the like. In other embodiments, aprocessed product includes, but is not limited to, a flour, meal, oil,starch, cereal, and the like produced from a harvested seed of theinvention, wherein said seed comprises a recombinant nucleic acidmolecule/nucleotide sequence of the invention.

Non-limiting examples of plants can include vegetable crops, includingartichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head,leaf, romaine), bok choy, malanga, melons (e.g., muskmelon, watermelon,crenshaw, honeydew, cantaloupe), cole crops (e.g., brussels sprouts,cabbage, cauliflower, broccoli, collards, kale, chinese cabbage, bokchoy) cardoni, carrots, napa, okra, onions, celery, parsley, chick peas,parsnips, chicory, peppers, potatoes, cucurbits (e.g., marrow, cucumber,zucchini, squash, pumpkin), radishes, dry bulb onions, rutabaga,eggplant (also called brinjal), salsify, escarole, shallots, endive,garlic, spinach, green onions, squash, greens, beet (sugar beet andfodder beet), sweet potatoes, swiss chard, horseradish, tomatoes,turnips, and spices; a fruit and/or vine crop such as apples, apricots,cherries, nectarines, peaches, pears, plums, prunes, cherry, quince,almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus,blueberries, boysenberries, cranberries, currants, loganberries,raspberries, strawberries, blackberries, grapes, avocados, bananas,kiwi, persimmons, pomegranate, pineapple, tropical fruits, pomes, melon,mango, papaya, and lychee; a field crop plant such as clover, alfalfa,evening primrose, meadow foam, corn/maize (field, sweet, popcorn), hops,jojoba, peanuts, rice, safflower, small grains (barley, oats, rye,wheat, etc.), sorghum, tobacco, kapok, a leguminous plant (beans,lentils, peas, soybeans), an oil plant (rape, mustard, poppy, olive,sunflower, coconut, castor oil plant, cocoa bean, groundnut),Arabidopsis, a fibre plant (cotton, flax, hemp, jute), lauraceae(cinnamon, camphor), or a plant such as coffee, sugar cane, tea, andnatural rubber plants; and/or a bedding plant such as a flowering plant,a cactus, a succulent and/or an ornamental plant, as well as trees suchas forest (broad-leaved trees and evergreens, such as conifers), fruit,ornamental, and nut-bearing trees, as well as shrubs and other nurserystock.

In some embodiments, a plant can be any plant species or plant varietiessusceptible to soybean cyst nematode infection including, but notlimited to, China pinks, edible beans, lespedeza, vetch (common, hairyor winter), lupine, clover (crimson, scarlet or alsike), sweetclover,birdsfoot trefoil, crownvetch, garden pea, cowpea, black-eyed pea,soybeans (wild and cultivated), black locust, honey locust, portulaca,Bells of Ireland, common chickweed, mousear chickweed, mullein,sicklepod, Digitalis penstemon, pokeweed, purslane, bittercress, RockyMountain beeplant, spotted geranium, toadflax, winged pigweed, Psoraleaspp., Cleome serrulata, vetch (American, Carolina or wood), burclover(Medicago minima), chick-weed (Cerastium vulgatum), dalea, Canadianmilkvetch, hemp sesbania, borage, canary bird flower, cup flower,caraway, Chinese lantern plant, blue gem viscaria, coralbell, Margaretdouble carnation, Rosa multiflora, pink queen, geranium (Geraniummaculatum), cup-flower, delphinium, foxglove, geum, common horehound,poppy, sage, snapdragon, beard-tongue (Penstemon digitalis), Desmodiumnudifolorum, D. marilandicum, D. viridiflorum, corn cockle, sweet basil,sweetpea, verbena, henbit (Lamium amplexicaule), purple deadnettle(Lamium purpureum), (field pennycress (Thlaspi arvense),shepherd's-purse (Capsella bursa-pastoris), hop clovers, beggars weed,tick clover, corn cockle, hogpeanut, milkpea, and wildbean(Strophostyles helvola).

In some particular embodiments, a transgenic plant of the inventionincludes, but is not limited to, a transgenic soybean plant, atransgenic sugar beet plant, a transgenic corn plant, a transgeniccotton plant, a transgenic canola plant, a transgenic wheat plant, or atransgenic rice plant. In other embodiments, a transgenic plant cell ofthe invention includes, but is not limited to, a transgenic soybeancell, a transgenic sugar beet cell, a transgenic corn cell, a transgeniccotton cell, a transgenic canola cell, a transgenic sugar cane cell, atransgenic wheat cell, or a transgenic rice cell.

As used herein, the term “plant part” includes but is not limited toembryos, pollen, ovules, seeds, leaves, flowers, branches, fruit,kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plantcells including plant cells that are intact in plants and/or parts ofplants, plant protoplasts, plant tissues, plant cell tissue cultures,plant calli, plant clumps, and the like. Further, as used herein, “plantcell” refers to a structural and physiological unit of the plant, whichcomprises a cell wall and also may refer to a protoplast. A plant cellof the invention can be in the form of an isolated single cell or can bea cultured cell or can be a part of a higher-organized unit such as, forexample, a plant tissue or a plant organ. A “protoplast” is an isolatedplant cell without a cell wall or with only parts of the cell wall.Thus, in some embodiments of the invention, a transgenic cell comprisinga nucleic acid molecule and/or nucleotide sequence of the invention is acell of any plant or plant part including, but not limited to, a rootcell, a leaf cell, a tissue culture cell, a seed cell, a flower cell, afruit cell, a pollen cell, and the like.

In some particular embodiments, the invention provides a transgenic seedproduced from a transgenic plant of the invention, wherein thetransgenic seed comprises a nucleic acid molecule/nucleotide sequence ofthe invention.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment. In some embodiments of the invention, a transgenic tissueculture or transgenic plant cell culture is provided, wherein thetransgenic tissue or cell culture comprises a nucleic acidmolecule/nucleotide sequence of the invention.

As used herein, a “plant organ” is a distinct and visibly structured anddifferentiated part of a plant such as a root, stem, leaf, flower bud,or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

The term “nematode plant pest” as used herein includes any nematodespecies that is a pest on a plant. Non-limiting examples of nematodepests include cyst nematodes (Heterodera spp.), especially the soybeancyst nematode (Heterodera glycines), root knot nematodes (Meloidogynespp.), lance nematodes (Hoplolaimus spp.), stunt nematodes(Tylenchorhynchus spp.), spiral nematodes (Helicotylenchus spp.), lesionnematodes (Pratylenchus spp.), sting nematodes (Belonoluimus spp.),reniform nematodes (Rotylenchulus reniformis), burrowing nematodes(Radopholus similis), Citrus nematode (Tylenchulus semipenetrans), andring nematodes (Criconema spp.).

“Introducing,” in the context of a nucleotide sequence of interest(e.g., the nucleotide sequences and nucleic acid molecules of theinvention), means presenting the nucleotide sequence of interest to theplant, plant part, and/or plant cell in such a manner that thenucleotide sequence gains access to the interior of a cell. Where morethan one nucleotide sequence is to be introduced these nucleotidesequences can be assembled as part of a single polynucleotide or nucleicacid construct, or as separate polynucleotide or nucleic acidconstructs, and can be located on the same or different transformationvectors. Accordingly, these polynucleotides can be introduced into plantcells in a single transformation event, in separate transformationevents, or, e.g., as part of a breeding protocol. Thus, the term“transformation” as used herein refers to the introduction of aheterologous nucleic acid into a cell. Transformation of a cell may bestable or transient. Thus, in some embodiments, a plant cell of theinvention is stably transformed with a nucleic acid molecule of theinvention. In other embodiments, a plant of the invention is transientlytransformed with a nucleic acid molecule of the invention.

“Transient transformation” in the context of a polynucleotide means thata polynucleotide is introduced into the cell and does not integrate intothe genome of the cell.

By “stably introducing” or “stably introduced” in the context of apolynucleotide introduced into a cell is intended the introducedpolynucleotide is stably incorporated into the genome of the cell, andthus the cell is stably transformed with the polynucleotide.

“Stable transformation” or “stably transformed” as used herein meansthat a nucleic acid is introduced into a cell and integrates into thegenome of the cell. As such, the integrated nucleic acid is capable ofbeing inherited by the progeny thereof, more particularly, by theprogeny of multiple successive generations. “Genome” as used herein alsoincludes the nuclear and the plastid genome, and therefore includesintegration of the nucleic acid into, for example, the chloroplastgenome. Stable transformation as used herein can also refer to atransgene that is maintained extrachromasomally, for example, as aminichromosome.

Transient transformation may be detected by, for example, anenzyme-linked immunosorbent assay (ELISA) or Western blot, which candetect the presence of a peptide or polypeptide encoded by one or moretransgene introduced into an organism. Stable transformation of a cellcan be detected by, for example, a Southern blot hybridization assay ofgenomic DNA of the cell with nucleic acid sequences which specificallyhybridize with a nucleotide sequence of a transgene introduced into anorganism (e.g., a plant). Stable transformation of a cell can bedetected by, for example, a Northern blot hybridization assay of RNA ofthe cell with nucleic acid sequences which specifically hybridize with anucleotide sequence of a transgene introduced into a plant or otherorganism. Stable transformation of a cell can also be detected by, e.g.,a polymerase chain reaction (PCR) or other amplification reactions asare well known in the art, employing specific primer sequences thathybridize with target sequence(s) of a transgene, resulting inamplification of the transgene sequence, which can be detected accordingto standard methods Transformation can also be detected by directsequencing and/or hybridization protocols well known in the art.

A nucleic acid of the invention (e.g., one or more of the nucleotidesequences of SEQ ID NOs:1-28, SEQ ID NOs:43-134, SEQ ID NOs:210-242, SEQID NOs:261-644, or a nucleotide sequence encoding one or morepolypeptides having the amino acid sequence of any one of SEQ IDNOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, SEQ ID NOs:665-1046)can be introduced into a cell by any method known to those of skill inthe art.

In some embodiments of the invention, transformation of a cell comprisesnuclear transformation. In other embodiments, transformation of a cellcomprises plastid transformation (e.g., chloroplast transformation).

Procedures for transforming plants are well known and routine in the artand are described throughout the literature. Non-limiting examples ofmethods for transformation of plants include transformation viabacterial-mediated nucleic acid delivery (e.g., via Agrobacteria),viral-mediated nucleic acid delivery, silicon carbide or nucleic acidwhisker-mediated nucleic acid delivery, liposome mediated nucleic aciddelivery, microinjection, microparticle bombardment,calcium-phosphate-mediated transformation, cyclodextrin-mediatedtransformation, electroporation, nanoparticle-mediated transformation,sonication, infiltration, PEG-mediated nucleic acid uptake, as well asany other electrical, chemical, physical (mechanical) and/or biologicalmechanism that results in the introduction of nucleic acid into theplant cell, including any combination thereof. General guides to variousplant transformation methods known in the art include Miki et al.(“Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) andRakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

Agrobacterium-mediated transformation is a commonly used method fortransforming plants, in particular, dicot plants, because of its highefficiency of transformation and because of its broad utility with manydifferent species. Agrobacterium-mediated transformation typicallyinvolves transfer of the binary vector carrying the foreign DNA ofinterest to an appropriate Agrobacterium strain that may depend on thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) PlantCell 5:159-169). The transfer of the recombinant binary vector toAgrobacterium can be accomplished by a triparental mating procedureusing Escherichia coli carrying the recombinant binary vector, a helperE. coli strain that carries a plasmid that is able to mobilize therecombinant binary vector to the target Agrobacterium strain.Alternatively, the recombinant binary vector can be transferred toAgrobacterium by nucleic acid transformation (Höfgen & Willmitzer (1988)Nucleic Acids Res. 16:9877).

Transformation of a plant by recombinant Agrobacterium usually involvesco-cultivation of the Agrobacterium with explants from the plant andfollows methods well known in the art. Transformed tissue is regeneratedon selection medium carrying an antibiotic or herbicide resistancemarker between the binary plasmid T-DNA borders.

Another method for transforming plants, plant parts and/or plant cellsinvolves propelling inert or biologically active particles at planttissues and cells. See, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006 and5,100,792. Generally, this method involves propelling inert orbiologically active particles at the plant cells under conditionseffective to penetrate the outer surface of the cell and affordincorporation within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the nucleic acid of interest.Alternatively, a cell or cells can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing one or more nucleic acids sought tobe introduced) also can be propelled into plant tis sue.

Thus, in particular embodiments of the invention, a plant cell can betransformed by any method known in the art and as described herein andintact plants can be regenerated from these transformed cells using anyof a variety of known techniques. Plant regeneration from plant cells,plant tissue culture and/or cultured protoplasts is described, forexample, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1,MacMilan Publishing Co. New York (1983)); and Vasil I. R. (ed.) (CellCulture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol.I (1984), and Vol. II (1986)). Methods of selecting for transformedtransgenic plants, plant cells and/or plant tissue culture are routinein the art and can be employed in the methods of the invention providedherein.

Likewise, the genetic properties engineered into the transgenic seedsand plants, plant parts, and/or plant cells of the invention describedabove can be passed on by sexual reproduction or vegetative growth andtherefore can be maintained and propagated in progeny plants. Generally,maintenance and propagation make use of known agricultural methodsdeveloped to fit specific purposes such as harvesting, sowing ortilling.

A nucleotide sequence therefore can be introduced into the plant, plantpart and/or plant cell in any number of ways that are well known in theart. The methods of the invention do not depend on a particular methodfor introducing one or more nucleotide sequences into a plant, only thatthey gain access to the interior of at least one cell of the plant.Where more than one nucleotide sequence is to be introduced, they can beassembled as part of a single nucleic acid construct, or as separatenucleic acid constructs, and can be located on the same or differentnucleic acid constructs. Accordingly, the nucleotide sequences can beintroduced into the cell of interest in a single transformation event,in separate transformation events, or, for example, in plants, as partof a breeding protocol.

Thus, in additional embodiments, the invention provides a method ofproducing a plant having increased resistance to infestation by anematode plant pest, the method comprising the steps of (a) crossing atransgenic plant of the invention with itself or another plant toproduce seed comprising a recombinant nucleic acid molecule or vector ofthe invention; and (b) growing a progeny plant from said seed to producea plant having increased resistance to infestation by nematode plantpests. In some embodiments, the method further comprises (c) crossingthe progeny plant of (b) with itself or another plant and (d) repeatingsteps (b) and (c) for an additional 0-7 (e.g., 0, 1, 2, 3, 4, 5, 6, 7)generations to produce a plant having increased resistance toinfestation by nematode plant pests.

In further embodiments, a method of producing a soybean plant havingincreased resistance to infestation by a nematode plant pest isprovided, the method comprising the steps of (a) crossing a transgenicsoybean plant of the invention with itself or another soybean plant toproduce soybean seed comprising a recombinant nucleic acid molecule orvector of the invention; and (b) growing a progeny soybean plant fromsaid seed to produce a soybean plant having increased resistance toinfestation by nematode plant pests. In some embodiments, the methodfurther comprises (c) crossing the progeny soybean plant of (b) withitself or another soybean plant and (d) repeating steps (b) and (c) foran additional 0-7 (e.g., 0, 1, 2, 3, 4, 5, 6, 7) generations to producea soybean plant having increased resistance to infestation by nematodeplant pests.

The invention further provides a plant crop comprising a plurality oftransgenic plants of the invention planted together in an agriculturalfield.

In addition, a method of improving the yield of a plant crop when saidplant crop is contacted with a nematode plant pest is provided, themethod comprising cultivating a plurality of plants comprising arecombinant nucleic acid molecule of the invention as the plant crop,wherein the plurality of plants of said plant crop have increasedresistance to nematode infection, thereby improving the yield of saidplant crop as compared to a control plant crop contacted with saidnematode plant pest, wherein the control plant crop is produced from aplurality of plants lacking said nucleic acid molecule. In someparticular embodiments of the invention, the crop is a soybean crop.

In some embodiments, a method of improving the yield of a crop when saidcrop is contacted with a nematode plant pest is provided, the methodcomprising contacting the nematode plant pest with an effective amountof a polypeptide of the invention or a nematicidal composition of theinvention, wherein the yield of the crop is improved as compared to aplant crop contacted with a nematode plant pest that has not beencontacted with said polypeptide and/or nematicidal composition. In someparticular embodiments of the invention, the crop is a soybean crop.

In still other embodiments, the invention further provides methods forcontrolling a nematode plant pest, methods of reducing the infectivityof a nematode plant pest toward a plant, methods of reducing infestationof a plant by a nematode plant pest, methods of reducing nematode cystdevelopment, and methods of reducing the growth of a nematode plant pestcomprising contacting the nematode plant pest with a composition of theinvention, wherein said composition comprises a recombinant nucleic acidmolecule, a nucleotide sequence, and/or a polypeptide of this invention.In some particular embodiments, the composition of the invention is atransgenic plant cell, transgenic plant or transgenic plant partcomprising and expressing a recombinant nucleic acid molecule/nucleotidesequence of the invention.

Accordingly, in one embodiment, the invention provides a method ofcontrolling a nematode plant pest, comprising contacting the nematodeplant pest with an effective amount of a polypeptide of the invention orcomposition thereof, thereby controlling the nematode plant pest ascompared to the control of a nematode plant pest which has not beencontacted with said polypeptide or composition thereof.

Thus, in a further embodiment, the invention provides a method ofcontrolling a nematode plant pest, comprising contacting the nematodeplant pest with a transgenic plant and/or a part thereof comprising arecombinant nucleic acid molecule of the invention, thereby controllingthe nematode plant pest as compared to the control of a nematode plantpest contacted with a control plant or plant part, said control plantlacking said recombinant nucleic acid molecule.

To “contact” a nematode plant pest with a polypeptide of the inventionand/or composition thereof or to “deliver” to a nematode plant pest apolypeptide of the invention and/or composition thereof means that thenematode plant pest comes into contact with, is exposed to, thepolypeptides of this invention and/or compositions thereof, resulting ina toxic effect on and control of the nematode (e.g., control, reducedinfectivity, reduced infestation, reduced cyst formation, reducedgrowth, and the like). A nematode plant pest can be contacted with apolypeptide of the invention or nematicidal composition of the inventionusing any art known method. For example, contacting includes but is notlimited to, (1) providing the polypeptide(s) of the invention in atransgenic plant, wherein the nematode eats (ingests) one or more partsof the transgenic plant, (2) in a protein composition(s) that can beapplied to the surface of a plant or plant part, for example, sprayedonto the plant surface, applied as a soil drench near the plant roots,or as a dip for a whole plant or parts thereof (e.g., roots) or (3) anyother art-recognized delivery system.

“Effective amount” refers to that concentration or amount of apolypeptide or nematicidal composition that inhibits or reduces theability of a nematode plant pest to survive, grow, feed and/orreproduce, or that limits nematode-related damage or loss in cropplants. Thus, in some embodiments of the invention, an “effectiveamount” can mean killing the nematode. In other embodiments, an“effective amount” does not mean killing the nematode.

The term “control” in the context of an effect on an organism (e.g.,nematode plant pest) means to inhibit or reduce, through a toxic effect,the ability of the organism to survive, grow, feed, and/or reproduce, orto limit damage or loss in crop plants that is related to the activityof the organism. To “control” an organism may or may not mean killingthe organism, although in some embodiments “control” means killing theorganism.

Thus, in particular embodiments, the overexpression of a nucleic acidmolecule of the invention in a plant results in the production of theencoded polypeptide, thereby conferring on a plant resistance to anematode plant pest. While not wishing to be bound by any particulartheory, the polypeptides of this invention may have a “direct toxic”effect on the nematodes or instead may be triggers for the production ofother proteins or metabolites or for one or more different pathways anyof which may exert a toxic effect on nematode plant pests.

In other embodiments of the invention, a method of reducing theinfectivity of a nematode plant pest to a plant is provided, the methodcomprising contacting the nematode plant pest with an effective amountof a polypeptide of the invention, thereby reducing the infectivity ofthe nematode plant pest to the plant as compared to the infectivity of anematode plant pest to which said polypeptide has not been delivered.

As used herein, “infect,” and “infectivity” means the ability of thenematode plant pest to infect, infest or parasitize a plant host.“Infest” and “infestation” refers to a pest nematode inhabiting oroverrunning a plant in numbers or quantities that are large enough to beharmful to the plant.

In some embodiments of the invention, a method of reducing theinfectivity of a nematode plant pest to a plant is provided, the methodcomprising contacting the nematode plant pest with a transgenic plantcomprising a recombinant nucleic acid molecule of the invention, therebyreducing the infectivity of the nematode plant pest as compared to anematode plant pest contacted with a control plant or plant part,wherein said control plant lacks said recombinant nucleic acid molecule.

In other embodiments, the invention provides a method of reducingnematode cyst development by a nematode plant pest on the roots of aplant, comprising contacting a nematode plant pest with an effectiveamount of the polypeptide of the invention, wherein nematode cystdevelopment by the nematode plant pest on the roots of said plant isreduced as compared to cyst development on the roots of a plant by anematode plant pest not contacted with said polypeptide.

In additional embodiments, a method of reducing nematode cystdevelopment by a nematode plant pest on roots of a plant is provided,the method comprising contacting a nematode plant pest with the roots ofa transgenic plant comprising a recombinant nucleic acid molecule of theinvention, wherein cyst development by the nematode plant pest on theroots of the transgenic plant is reduced as compared cyst development onthe roots of a control plant lacking said recombinant nucleic acidmolecule.

In other embodiments of the invention, a method of reducing the growthof a nematode plant pest population is provided, the method comprisingcontacting the nematode plant pest population with an effective amountof a polypeptide of the invention, wherein the growth of a nematodeplant pest population is reduced as compared to the growth of a controlnematode plant pest population not contacted with the polypeptide.

In still other embodiments, a method of reducing the growth of anematode plant pest population is provided, the method comprisingcontacting the nematode plant pest population with a transgenic plantcomprising a recombinant nucleic acid molecule of the invention, whereinthe growth of a nematode plant pest population is reduced as compared tothe growth of a nematode plant pest population contacted with a controlplant or plant part, said control plant or plant part lacking therecombinant nucleic acid molecule.

Thus, when a transgenic plant comprising a recombinant nucleic acidmolecule of the invention, or a part thereof, is exposed to or broughtinto contact with a nematode plant pest such that the nematode feeds onor otherwise contacts the transgenic plant or part thereof, the abilityof the nematode plant pest to survive, grow, feed, and/or reproduce inassociation with a plant is inhibited or reduced, thereby controllingthe nematode/nematode population and/or reducing the ability of thenematode plant pest to infect or infest a plant or produce cysts on aplant. Additionally, one or more polypeptides of the invention orcompositions comprising one or more polypeptides of the invention can beused directly to control or reduce the growth of a nematode plant pest,thereby reducing the ability of the nematode plant pest to infect orinfest a plant or produce cysts on a plant.

The invention will now be described with reference to the followingexamples. It should be appreciated that these examples are not intendedto limit the scope of the claims to the invention, but are ratherintended to be exemplary of certain embodiments. Any variations in theexemplified methods that occur to the skilled artisan are intended tofall within the scope of the invention.

EXAMPLES Example 1: Construction of Expression Cassettes for Hairy RootTransformation

At least one nucleic acid of the invention comprising a nucleotidesequence of any one of the nucleotide sequences of SEQ ID NOs:1-28, SEQID NOs:43-134, SEQ ID NOs:210-242, SEQ ID NOs:261-644, or a nucleotidesequence encoding any one of the polypeptides having the amino acidsequences of SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260,SEQ ID NOs:665-1046, is cloned into an expression cassette having thebasic structure from 5′ to 3′ of: 5′-promoter-nucleic acid of theinvention-terminator-3′. Expression cassettes may also compriseenhancers, introns, leader sequences and the like. One such expressioncassette has the structure: prActin2 (including Act2intron):cEVO18010081 (SEQ ID NO:24):tNOS or prActin2 (including Act2intron):cEVO18010044 (SEQ ID NO:224):tNOS. Other nucleic acids of theinvention can be substituted for the cEVO18010081 or EV018010044 codingsequence to create different expression cassettes. The expressioncassette is then cloned into a binary expression vector to create ahairy root (HR) transformation vector. A cEVO18010081 (SEQ ID NO:24) orcEVO18010044 (SEQ ID NO: 224) HR transformation vector was created bycloning the cEVO18010081 (SEQ ID NO:24) or cEVO18010044 (SEQ ID NO: 224)expression cassette, respectively, and a second expression cassetteencoding a scorable marker into a binary vector. As an example, theresulting HR transformation vector 20844 comprising cEVO18010081 (SEQ IDNO:24) is shown in FIG. 1.

Example 2: Expression in Transgenic Soybean Roots

The binary expression vector described in Example 1 containing a nucleicacid of the invention and an empty vector (without a nucleic acid of theinvention) shown in FIG. 2 was transformed into soybean roots to testthe binary vector's ability to express a protein that is capable ofreducing soybean cyst nematode (SCN) cysts. Soybean cultivar Williams 82was used as the germplasm for the hairy root transformation. Soybeanseeds were germinated on 1% agar containing 0.5% sucrose in Petri dishesat approximately 27° C. for 5 days. The cotyledons were then cut off theseedlings, and the wounded surface was inoculated with cultures of anAgrobacterium rhizogenes strain (e.g., K599) carrying the binaryexpression vector or empty vector. The cotyledons were placed on 1% agarfor about 6 days and then transferred onto selection media. In about twoweeks, independent transgenic hairy root events induced from thecotyledons were harvested and transferred onto culture media, andcultured in the darkness at about 27° C. Narayanan et al. (Crop Science39, 1680-1686 (1999)) indicates that SCN resistance phenotypes in awhole soybean plant are preserved in transgenic hairy roots, thereforethe transgenic hairy root system is useful for evaluating candidate SCNresistance genes and predicting activity in whole soybean plants.

Approximately two weeks after transfer onto the culture plates, thetransformed hairy roots were inoculated with surface-sterilized J2 stagesoybean cyst nematodes (SCN J2) and the roots were grown in darkness atabout 27° C., which allows cyst formation on the hairy root events. Onemonth after nematode inoculation, the number of cysts were determinedfor the roots expressing the polynucleotides of SEQ ID NOs:15-28, 31,35, 225, 227, 228, 230-234, or 238-240 (i.e., producing the polypeptidesof SEQ ID NOs: 29-38, 40-42, 52, 243, 244, 246, 248-252, 25, or 256-259)or for roots expressing the polynucleotides of SEQ ID NOs:1047-1062 andfor roots expressing the empty vector (as a negative control). Theexperiments were repeated at least one time.

The results of the experiments are shown in Tables 1-25 below.

TABLE 1 Average cyst Number of Standard number hairy root errorPlasmid_ID Nucleotide sequence (Avg) events (n) (SE) SCNBHR10 SEQ ID NO:15 22.1 17 2.5 SCNBHR25 SEQ ID NO: 21 24 3 5.6 SCNBHR52 SEQ ID NO: 2312.2 11 3.4 SCNBHR81 SEQ ID NO: 24 33.6 14 4 SCNBHRCK Empty Vector 21.211 3

TABLE 2 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR18 SEQ ID NO: 1810.8 16 2.4 SCNBHR21 SEQ ID NO: 20 6.4 18 1.2 SCNBHR25 SEQ ID NO: 2110.5 13 1.1 SCNBHR36 SEQ ID NO: 22 12 8 1.7 SCNBHR52 SEQ ID NO: 23 2 1SCNBHR84 SEQ ID NO: 27 14.1 20 1.4 SCNBHRCK Empty Vector 13.3 9 1.7

TABLE 3 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR15 SEQ ID NO: 1715 9 3.6 SCNBHR82 SEQ ID NO: 25 16.9 15 2.5 SCNBHRCK Empty Vector 10.412 1.6

TABLE 4 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR10 SEQ ID NO: 1515 2 2 SCNBHR15 SEQ ID NO: 17 12.3 7 2.4 SCNBHR36 SEQ ID NO: 22 14.5 102.6 SCNBHR52 SEQ ID NO: 23 6 1 SCNBHR81 SEQ ID NO: 24 5 3 0 SCNBHR82 SEQID NO: 25 8 1 SCNBHRCK Empty Vector 20.5 2 2.5

TABLE 5 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR11 SEQ ID NO: 1627.6 16 2.3 SCNBHR19 SEQ ID NO: 19 24.1 16 3 SCNBHR83 SEQ ID NO: 26 17.814 2.2 SCNBHR88 SEQ ID NO: 28 21.6 13 2.7 SCNBHRCK Empty Vector 18.5 102.5

TABLE 6 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR10 SEQ ID NO: 1543 1 SCNBHR15 SEQ ID NO: 17 41.3 3 12.4 SCNBHR21 SEQ ID NO: 20 27.9 72.9 SCNBHR36 SEQ ID NO: 22 32 3 11.9 SCNBHR52 SEQ ID NO: 23 42.7 7 4.2SCNBHR81 SEQ ID NO: 24 19 1 SCNBHR83 SEQ ID NO: 26 31.5 2 1.5 SCNBHRCKEmpty Vector 60.8 5 4

TABLE 7 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR40 SEQ ID NO: 104734.5 12 6 SCNBHR44 SEQ ID NO: 225 29.6 12 3.5 SCNBHR47 SEQ ID NO: 22729.1 13 3.5 SCNBHR50 SEQ ID NO: 1048 31.1 13 3.2 SCNBHR55 SEQ ID NO:1049 36.5 15 3.8 SCNBHR57 SEQ ID NO: 1050 40.1 14 3.2 SCNBHR60 SEQ IDNO: 232 20.7 11 3.2 SCNBHR65 SEQ ID NO: 1051 33.8 11 5.9 SCNBHRCK EmptyVector 39.9 11 4.3

TABLE 8 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR43 SEQ ID NO: 105270.9 7 4.8 SCNBHR48 SEQ ID NO: 228 23.3 7 9.7 SCNBHR52 SEQ ID NO: 2324.8 4 6.2 SCNBHR71 SEQ ID NO: 1053 66.6 8 11.1 SCNBHR72 SEQ ID NO: 105471.6 9 5.2 SCNBHR79 SEQ ID NO: 1055 108.8 4 32.6 SCNBHR91 SEQ ID NO:1056 48 4 12.2 SCNBHRCK Empty Vector 72.7 3 22.3

TABLE 9 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR58 SEQ ID NO: 105743.6 8 6 SCNBHR59 SEQ ID NO: 231 97 13 8.1 SCNBHR86 SEQ ID NO: 1058 58.47 15.2 SCNBHR89 SEQ ID NO: 1059 45.5 6 7 SCNBHRCK Empty Vector 62.9 109.4

TABLE 10 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR43 SEQ ID NO:1052 44.9 10 5.1 SCNBHR79 SEQ ID NO: 1055 44.7 12 4.2 SCNBHR91 SEQ IDNO: 1056 30.7 3 9.8 SCNBHRCK Empty Vector 46.8 10 5.8

TABLE 11 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR71 SEQ ID NO:1053 65 7 11 SCNBHR72 SEQ ID NO: 1054 53.7 8 8.9 SCNBHR91 SEQ ID NO:1056 46 3 7.6 SCNBHRCK Empty Vector 65.4 8 7.4

TABLE 12 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR48 SEQ ID NO: 22834.6 8 7.4 SCNBHR61 SEQ ID NO: 233 53.2 6 5.9 SCNBHR73 SEQ ID NO: 238 447 8 SCNBHR77 SEQ ID NO: 239 34.8 6 6.4 SCNBHRCK Empty Vector 78 9 10.6

TABLE 13 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR68 SEQ ID NO:1060 46.2 6 10.7 SCNBHR70 SEQ ID NO: 1061 53.7 3 12.8 SCNBHR80 SEQ IDNO: 240 34.5 2 7.5 SCNBHR85 SEQ ID NO: 1062 45 2 5 SCNBHRCK Empty Vector55.2 6 6.3

TABLE 14 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR15 SEQ ID NO: 1745.8 12 3.6 SCNBHR21 SEQ ID NO: 20 51.1 14 4.1 SCNBHR36 SEQ ID NO: 2250.4 5 4.9 SCNBHR52 SEQ ID NO: 23 45.5 13 4.1 SCNBHR66 SEQ ID NO: 236 627 11.7 SCNBHR77 SEQ ID NO: 239 57.2 12 6 SCNBHR81 SEQ ID NO: 24 30.2 122.6 SCNBHRCK Empty Vector 65.7 17 5.8

TABLE 15 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR10 SEQ ID NO: 1563 11 3.9 SCNBHRCK Empty Vector 97.4 14 8.3

TABLE 16 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR59 SEQ ID NO: 231398.1 19 17.15435 SCNBHRCK Empty Vector 312.4 11 29.17074

TABLE 17 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR43 SEQ ID NO:1052 84.5 14 6.8 SCNBHR58 SEQ ID NO: 1057 85.4 9 5.9 SCNBHRCK EmptyVector 89.8 11 4.2

TABLE 18 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR54 SEQ ID NO: 23069.9 13 4.9 SCNBHR61 SEQ ID NO: 233 66.3 13 5.1 SCNBHR73 SEQ ID NO: 23896.5 13 11.6 SCNBHRCK Empty Vector 103.2 13 9.6

TABLE 19 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR60 SEQ ID NO: 23282.6 9 5.8 SCNBHRCK Empty Vector 145.8 10 17.8

TABLE 20 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR80 SEQ ID NO: 24065.3 19 4.6 SCNBHRCK Empty Vector 61 14 5.4

TABLE 21 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR47 SEQ ID NO: 22757.5 13 3.7 SCNBHR53 SEQ ID NO: 229 61.9 16 3.4 SCNBHR92 SEQ ID NO: 24285.3 15 5.6 SCNBHRCK Empty Vector 81.5 16 5

TABLE 22 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR83 SEQ ID NO: 2675.7 15 5.9 SCNBHRCK Empty Vector 104.7 14 8.

TABLE 23 Plasmid_ID Nucleotide sequence Avg n SE SCNBHR44 SEQ ID NO: 22597.9 20 5 SCNBHR60 SEQ ID NO: 232 72.3 12 5.8 SCNBHR63 SEQ ID NO: 234102.5 19 5.7 SCNBHR82 SEQ ID NO: 25 109.8 17 3.9 SCNBHRCK Empty Vector109.6 16 4.8

TABLE 24 Plasmid_ID Nucleotide sequence Cysts n SE SCNBHR77 SEQ ID NO:239 63 18 3.3 SCNBHRCK Empty Vector 71 13 4.4

TABLE 25 Vector Nucleotide sequence Cysts n SE SCNBHR54 SEQ ID NO: 23036 19 2.3 SCNBHR73 SEQ ID NO: 238 40 11 1.5 SCNBHRCK Empty Vector 33 192.3

The results of these experiments show that the number of cysts formed onsoybean roots expressing at least a polynucleotide sequence having thenucleotide sequence of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:227, SEQ IDNO:228, SEQ ID NO:230, SEQ ID NO:232 and/or SEQ ID NO:233 wassignificantly lower than on transgenic soybean roots comprising theempty vector control. Those skilled in the art would understand that thegenomic sequences and/or mRNA plus UTR and ORF sequences correspondingto the above SEQ ID NOs as provided herein could also be used to reducenematode parasitism (e.g., SEQ ID NOs:1, 3, 6, 8, 9, 10, 12, 43, 45, 48,50, 51, 53, 211, 212, 214, 216, 217).

Polynucleotides having substantial sequence identity (e.g., at least 80%identity) to the polynucleotides shown above as reducing the number ofcysts on soybean roots may also be useful for reducing nematodeinfestation, cyst number, and the like, in plants. Non-limiting examplesof polynucleotides having substantial identity to the nucleotidesequence of SEQ ID NO:15 include the nucleotide sequences of SEQ ID NOs:56-60; to the nucleotide sequence of SEQ ID NO:17 includes thenucleotide sequence of SEQ ID NO:63; to the nucleotide sequence of SEQID NO:20 includes the nucleotide sequences of SEQ ID NO:66 and/or SEQ IDNO:67; to the nucleotide sequence of SEQ ID NO:22 includes thenucleotide sequences of SEQ ID NOs:68-112; to the nucleotide sequence ofSEQ ID NO:23 includes the nucleotide sequences of SEQ ID NOs:113-118; tothe nucleotide sequence of SEQ ID NO:24 includes the nucleotide sequenceof SEQ ID NO:119; to the nucleotide sequence of SEQ ID NO:26 includesthe nucleotide sequences of SEQ ID NOs:120-124; to the nucleotidesequence of SEQ ID NO:227 includes the nucleotide sequences of SEQ IDNOs:226, 389-398; to the nucleotide sequence of SEQ ID NO:228 includesthe nucleotide sequences of SEQ ID NOs:399-401; to the nucleotidesequence of SEQ ID NO:230 includes the nucleotide sequences of SEQ IDNOs:408-633; and/or to the nucleotide sequence of SEQ ID NO:232 includesthe nucleotide sequences of SEQ ID NOs:637-642.

Example 3: Construction of Expression Cassettes and Vectors for SoybeanTransformation

The expression cassettes described in Example 1 are used in soybeantransformation experiments or different expression cassettes areconstructed. At least one nucleic acid comprising a nucleotide sequenceselected from the nucleotide sequences of any one of SEQ ID NOs:1-28,43-134, 210-242, 261-664 or nucleotide sequences encoding thepolypeptide having the amino acid sequence of any one of SEQ IDNOs:29-42, 135-209, 243-260, 665-1046, is cloned into an expressioncassette and the expression cassette cloned into a binary vector for thegeneration of transgenic soybean plants. The genetic material to betransferred to the soybean plant is cloned between the left border andthe right border of the binary vector. One such expression cassette hasthe structure: eFMV:e35S:prAct2 (including Act2 intron): cEVO18010081(SEQ ID NO:24):tNOS. The cEVO18010081 (SEQ ID NO:24) expression cassetteand a second expression cassette encoding a selectable marker are clonedinto a binary vector to create 20944 EVO shown in FIG. 3. Anotherexemplary expression cassette has the structure: eFMV:e35S:prAct2(including Act2 intron): cEVO18010044 (SEQ ID NO:224):tNOS.

The binary vector comprising an expression cassette described above isintroduced into an Agrobacterium tumefaciens strain (e.g., EHA101),using electroporation. Single bacterial colonies containing the binaryvector are selected to confirm the presence of intact vector and usedfor further experiments.

Example 4: Production of Transgenic Soybean

Transformation of soybean to produce transgenic soybean plants wasaccomplished using targets prepared from germinated seeds of varietyWilliams 82 via Agrobacterium tumefaciens-mediated transformation asdescribed here. Mature soybean seeds were harvested, dried andsterilized with chlorine gas. Sterilized seeds were placed in laminarflow hoods for 2 weeks before germination. Seeds were placed ongerminated media for 15 to 40 hours for germination. Explants wereprepared as described in Khan (US patent application 20040034889) usinggerminated seeds by removing hypocotyls, one cotyledon and primary leafprimordial. The explants were then wounded by gentle wounding at thecotyledonary nodal region and also apical regions. Explants were theninfected with Agrobacterium strain EHA101 containing appropriate binaryvector. Infected explants were co-cultured in co-cultivation media asdescribed in Hwang et al 2008 (WO08112044). Excess A. tumefacienssuspension was then removed by aspiration and explants were moved toplates containing a non-selective co-culture medium. Explants wereco-cultured with the remaining A. tumefaciens at 23° C. for 4 days inthe dark. Explants were then transferred to recovery medium supplementedwith an antibiotics mixture consisting of ticarcillin (75 mg/L),cefotaxime (75 mg/L) and vancomycin (75 mg/1) and incubated in the darkfor seven days as described in Hwang et al 2008 (WO08112044). Explantswere then transferred to regeneration medium containing glyphosate (75to 100 uM) and a mixture of antibiotics consisting of ticarcillin (75mg/L), cefotaxime (75 mg/L) and vancomycin (75 mg/1) to inhibit and killA. tumefaciens. Shoot elongation was carried out in elongation mediacontaining glyphosate (50 uM). The EPSPS gene was used as a selectablemarker during the transformation process. Regenerated plantlets weretransplanted to soil as described in Que et al (WO08112267) and testedfor the presence of both EPSPS marker gene and spectinomycin resistance(Spec) sequences by TaqMan PCR analysis (Ingham et al., 2001). Thisscreen allows for the selection of transgenic events that carry theT-DNA and are free of vector backbone DNA. Plants positive for EPSPSgene sequences and negative for the Spec gene were transferred to thegreenhouse for analysis of miRNA expression seed setting. Using thismethod, genetic elements within the left and right border regions of thetransformation plasmid are efficiently transferred and integrated intothe genome of the plant cell, while genetic elements outside theseborder regions are generally not transferred.

When the roots are about 2-3 inches, they are transplanted into 1-gallonpots using Fafard #3 soil and 30 grams of incorporated Osmocote Plus15-9-12. They are watered in thoroughly and placed in the cubicle underflorescent lighting set to a 16-hour day. The temperatures are about 85°F. (29.4° C.) during the day and about 70° F. (21° C.) at night. Plantsare watered at least once daily.

The plants remain in the cubicle until secondary Taqman sampling hasbeen performed, typically 1-2 weeks. The plants are then placed on anautomatic drip watering system and watered twice daily. A cage is placedover the plant, and it may be pruned very lightly if needed. Thelighting is a combination of Metal Halide and Sodium Vapor fixtures with400- and 1000-watt bulbs with a 10-hour day period. The outside wall isdarkened to keep out light that would extend the day length.Temperatures are set at about 79° F. (26° C.) during the day and about70° F. (21° C.) at night. The humidity is ambient.

The plants are maintained in this manner until pods reach maturity,approximately 100 days based on the date of the Taqman selection. Thepods are then harvested, placed in a paper bag, air-dried for about2-days, and then machine dried at about 80° F. (27° C.) for 2-additionaldays. The pods are shelled and the T1 seeds are harvested and stored atabout 4° C. until further testing.

Wild type Williams 82 or null segregants of the T1 generation are usedas a control in the SCN assay. Alternatively, a control in the SCN assaycan be a plant transformed with an empty vector such as shown in FIG. 4(i.e., an identical expression cassette but without a nucleotidesequence of SEQ ID NOs:1-28, SEQ ID NOs: 43-134, SEQ ID NOs:210-242, orSEQ ID NOs:261-644, and/or a nucleotide sequence encoding one or morepolypeptides having the amino acid sequence of SEQ ID NOs: 29-42, SEQ IDNOs:135-209, SEQ ID NOs:243-260, or SEQ ID NOs:665-1046.

Example 5: Evaluation of Cyst Formation in the Transformed SoybeanPlants

Soybean plants transformed with the expression cassette harboring atleast one nucleotide sequence of any one of the nucleotide sequences ofSEQ ID NOs:1-28, SEQ ID NOs: 43-134, SEQ ID NOs:210-242, SEQ IDNOs:261-644, or a nucleotide sequence encoding any one of thepolypeptides having the amino acid sequence of any one of SEQ ID NOs:29-42, SEQ ID NOs:135-209, SEQ ID NOs:243-260, or SEQ ID NOs:665-1046are inoculated with J2 stage soybean cyst nematodes (SCN J2). 3-week oldtransgenic T1 generation soybean seedlings grown in germination pouchesindividually are inoculated with SCN J2 suspension at the level of 500J2 per plant. The soybean plants were cultured at 27° C. in a growthchamber with 16 hours per day of light period.

One month after nematode inoculation, the number of cysts is determinedfor both the transgenic soybean plants comprising the at least onenucleotide sequence as set forth above and for the null segregants(plants not having a nucleic acid of the invention) from the same T0parents.

Example 6. Evaluation of the Role of Selected Recombinant Nucleic Acidsin Resistance and/or Tolerance to Nematodes

To validate the role of selected polynucleotides of the invention inplant resistance and or tolerance to nematodes the selectedpolynucleotides were over-expressed in plants, as follows.

Cloning strategy Selected polynucleotides having a nucleotide sequenceof SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:228, or SEQ ID NO:1047 were cloned into binaryvectors for the generation of transgenic plants.

For cloning, the full-length open reading frame (ORF) was firstidentified. In case of ORF-EST clusters and in some cases alreadypublished mRNA sequences were analyzed to identify the entire openreading frame by comparing the results of several translation algorithmsto known proteins from other plant species. To clone the full-lengthcDNAs, reverse transcription (RT) followed by polymerase chain reaction(PCR; RT-PCR) was performed on total RNA extracted from roots, leaves,flowers, siliques or other plant tissues, growing under normal anddifferent treated conditions. Total RNA was extracted using methods wellknown in the art. Production of cDNA and PCR amplification was performedusing standard protocols, which are well known to those skilled in theart (See, e.g., Sambrook J., E. F. Fritsch, and T. Maniatis. 1989.Molecular cloning: a laboratory manual, 2nd Ed. Cold Spring HarborLaboratory Press, New York). PCR products were purified using PCRpurification kit (Qiagen). In those instances where the entire codingsequence was not identified, RACE kit from Invitrogen (RACE=RapidAmplification of cDNA Ends) was used to access the full cDNA transcriptof the gene from the RNA samples described above. RACE products werecloned into high copy vector followed by sequencing or directlysequenced. The information from the RACE procedure was used for cloningof the full length ORF of the corresponding genes.

When genomic DNA was cloned, the genes were amplified by direct PCR ongenomic DNA extracted from leaf tissue using the DNAeasy kit (QiagenCat. No. 69104). Typically, 2 sets of primers were synthesized for theamplification of each gene from a cDNA or a genomic sequence; anexternal set of primers and an internal set (nested PCR primers). Whenneeded, an additional primer (or two) of the nested PCR primers wasused.

To facilitate the cloning of the cDNAs/genomic sequences, an 8-12 bpextension was added to the 5′ of each primer. The primer extensionincludes an endonuclease restriction site. The restriction sites wereselected using two parameters: (a) the site does not exist in the cDNAsequence, and (b) the restriction sites in the forward and reverseprimers are designed such that the digested cDNA is inserted in thesense formation into the binary vector that is utilized fortransformation.

Each digested PCR product was inserted into a high copy vector pUC19(New England BioLabs Inc) or into plasmids originating from this vector.In some cases, the undigested PCR product can be inserted into pCR-BluntII-TOPO (Invitrogen).

Sequencing of the amplified PCR products was performed, using ABI 377sequencer (Amersham Biosciences Inc). In some cases, after confirmingthe sequences of the cloned genes, the cloned cDNA was introduced into amodified pGI binary vector containing the At6669 promoter (SEQ IDNO:1063) via digestion with appropriate restriction endonucleases. Theinsert is then followed by single copy of the NOS terminator (Vancanneytet al. Molecular Genetics and Genomics 220, 245-50, 1990). The digestedproducts and the linearized plasmid vector were ligated using T4 DNAligase enzyme (Roche, Switzerland). High copy plasmids containing thecloned genes were digested with the restriction endonucleases (NewEngland BioLabs Inc) according to the sites designed in the primers andcloned into binary vectors.

Several DNA sequences of the selected genes were synthesized by acommercial supplier GeneArt (www(dot)geneart(dot)com). Synthetic DNA wasdesigned in silico. Suitable restriction enzymes sites were added to thecloned sequences at the 5′ end and at the 3′ end to enable later cloninginto the pQFNc binary vector downstream of the At6669 promoter

Binary vectors used for cloning: The plasmid pPI was constructed byinserting a synthetic poly-(A) signal sequence, originating from pGL3basic plasmid vector (Promega, Acc No U47295; bp 4658-4811) into theHindIII restriction site of the binary vector pBI101.3 (Clontech, Acc.No. U12640). pGI (pBXYN) is similar to pPI, but the original gene in thebackbone, the GUS gene, was replaced by the GUS-Intron gene followed bythe NOS terminator. pGI was used in the past to clone the polynucleotidesequences, initially under the control of 35S promoter (Odell et al.Nature 313, 810-812 (28 Feb. 1985)).

The modified pGI vectors (pQXNc (FIG. 7); or pQFN (FIG. 6), pQFNc (FIG.6) or pQYN 6669 (FIG. 5) are modified versions of the pGI vector inwhich the cassette was inverted between the left and right borders sothe gene and its corresponding promoter are close to the right borderand the NPTII gene is close to the left border.

At6669, the Arabidopsis thaliana promoter sequence (SEQ ID NO:1063) wasinserted in the modified pGI binary vector, upstream to the clonedgenes, followed by DNA ligation and binary plasmid extraction frompositive E. coli colonies, as described above.

Colonies were analyzed by PCR using the primers covering the insertwhich were designed to span the introduced promoter and gene. Positiveplasmids were identified, isolated and sequenced.

For cloning of each gene at least 2 primers were used, forward andreverse. In some cases, four primers were used: external forward,external reverse, nested forward or nested reverse. The genes werecloned from the indicated organism, except for the genes that weresynthetically produced by GeneArt.

Example 7. Producing Transgenic Arabidopsis Plants Expressing SelectedPolynucleotides

Production of Agrobacterium tumefaciens cells harboring the binaryvectors according to some embodiments of the invention. Each of thebinary vectors described in Example 6 above was used to transformAgrobacterium cells. An additional binary construct, having only theAt6669 promoter was used as negative control. The binary vectors wereintroduced to Agrobacterium tumefaciens GV301, or LB4404 competent cells(about 10⁹ cells/mL) by electroporation. The electroporation wasperformed using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes(Biorad) and EC-2 electroporation program (Biorad). The treated cellswere cultured in LB liquid medium at 28° C. for 3 hours, then platedover LB agar supplemented with gentamycin (50 mg/L; for Agrobacteriumstrains GV301) or streptomycin (300 mg/L; for Agrobacterium strainLB4404) and kanamycin (50 mg/L) at 28° C. for 48 hours. Agrobacteriumcolonies, which were developed on the selective media, were furtheranalyzed by PCR using the primers designed to span the inserted sequencein the pPI plasmid. The resulting PCR products were isolated andsequenced to verify that the correct polynucleotide sequences of theinvention were properly introduced to the Agrobacterium cells.

Preparation of Arabidopsis plants for transformation—Arabidopsisthaliana var Columbia (T₀ plants) were transformed according to thefloral dip procedure (Clough et al. (1998) Plant J. 16(6): 735-43; andDesfeux et al. (2000) Plant Physiol. 123(3): 895-904) with minormodifications. Briefly, Arabidopsis thaliana Columbia (Col0) T₀ plantswere sown in 250 ml pots filled with wet peat-based growth mix. The potswere covered with aluminum foil and a plastic dome, kept at 4° C. for3-4 days, then uncovered and incubated in a growth chamber at 18-24° C.under 16/8 hours light/dark cycles. The T₀ plants were ready fortransformation six days before anthesis.

Preparation of the Agrobacterium carrying the binary vectors totransformation into Arabidopsis plants—Single colonies of Agrobacteriumcarrying the binary vectors harboring the genes of some embodiments ofthe invention were cultured in LB medium supplemented with kanamycin (50mg/L) and gentamycin (50 mg/L). The cultures were incubated at 28° C.for 48 hours under vigorous shaking and centrifuged at 4000 rpm for 5minutes. The pellets comprising Agrobacterium cells were resuspended ina transformation medium which contains half-strength (2.15 g/L)Murashige-Skoog (Duchefa); 0.044 μM benzylamino purine (Sigma); 112 μg/LB5 Gambourg vitamins (Sigma); 5% sucrose; and 0.2 ml/L Silwet L-77 (OSISpecialists, CT) in double-distilled water, at pH of 5.7.

Transformation of Arabidopsis plants with theAgrobacterium—Transformation of T₀ plants was performed by invertingeach plant into an Agrobacterium suspension such that the above groundplant tissue is submerged for 3-5 seconds. Each inoculated T₀ plant wasimmediately placed in a plastic tray, then covered with clear plasticdome to maintain humidity and was kept in the dark at room temperaturefor 18 hours to facilitate infection and transformation. Transformed(transgenic) plants were then uncovered and transferred to a greenhousefor recovery and maturation. The transgenic T₀ plants were grown in thegreenhouse for 3-5 weeks until siliques are brown and dry, then seedswere harvested from plants and kept at room temperature until sowing.

Generation of T1 and T2 transgenic plants—For generating T₁ and T₂transgenic plants harboring the genes, seeds collected from transgenicT₀ plants were surface-sterilized by soaking in 70% ethanol for 1minute, followed by soaking in 5% sodium hypochlorite and 0.05% tritonfor 5 minutes. The surface-sterilized seeds were thoroughly washed insterile distilled water then placed on culture plates containinghalf-strength Murashig-Skoog (Duchefa); 2% sucrose; 0.8% plant agar; 50mM kanamycin; and 200 mM carbenicylin (Duchefa). The culture plates wereincubated at 4° C. for 48 hours then transferred to a growth room at 25°C. for an additional week of incubation. Vital T₁ Arabidopsis plantswere transferred to a fresh culture plates for another week ofincubation. Following incubation the T₁ plants were removed from cultureplates and planted in growth mix contained in 250 ml pots. Thetransgenic plants were allowed to grow in a greenhouse to maturity.Seeds harvested from T₁ plants were cultured and grown to maturity as T₂plants under the same conditions as used for culturing and growing theT₁ plants.

Example 8. Evaluation of Transgenic Arabidopsis for Reduced Infection byNematodes

The binary expression vector described above, pQFN or pQFNc includingthe At6669 promoter containing at least one nucleic acid of theinvention or an empty vector (without a nucleic acid of the invention)were transformed into Arabidopsis to test the ability of the binaryvector to express a protein that is capable of reducing sugar beet cystnematode (BCN) cysts. Arabidopsis cultivar Columbia-0 was used as thegermplasm for transformation. Arabidopsis seeds were germinated on 3%phytagel containing 0.5% sucrose in 1.5 ml tubes at approximately 25° C.for 10 days. Individual plants, 5 to 10 plants per event and 3 to 7events per SEQ ID, were then transferred to 0.25 liter pots containingsand and grown for additional 10 days in the green house. The pots werethen inoculated with J2 stage sugar beet cyst nematodes (BCN J2) andplants were grown in green house at about 25° C., which allows cystformation on the root of the plants. One month after nematodeinoculation, the number of cysts was determined for both the rootsexpressing at least one of the polynucleotides of SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:228, or SEQ ID NO:1047 and the roots expressing the empty vector (asa negative control). The experiment was repeated at least one time.

TABLE 26 Genes showing reduced plant infection to nematodes. Nematodefemale cysts per plant % of SEQ ID NO Event # Mean SE (±) p-Valuecontrol Empty vector control Mix135 9.89 1.20 1.00 — SEQ ID NO: 104761990.12 2.90 0.75 <.0001 29 SEQ ID NO: 1047 61991.13 7.10 1.67 0.34 72SEQ ID NO: 1047 61995.2 6.10 0.74 0.08 62 SEQ ID NO: 228 60025.11 1.000.52 <.0001 10 SEQ ID NO: 228 62025.7 6.50 0.79 0.15 66 SEQ ID NO: 22862026.11 6.67 1.81 0.22 67 SEQ ID NO: 16 62118.11 3.09 0.59 <.0001 31SEQ ID NO: 16 62118.12 3.10 0.66 0.00 31 SEQ ID NO: 16 62121.6 3.30 0.600.00 33 SEQ ID NO: 16 62122.4 3.80 1.28 0.00 38 SEQ ID NO: 16 62392.42.50 0.91 <.0001 25

TABLE 27 Genes showing reduced plant infection to nematodes. Nematodefemale cysts per plant % of SEQ ID NO Event # Mean SE (±) p-Valuecontrol Empty vector control mix138-b 11.5 0.63 1 — SEQ ID NO: 1861888.3 5.5556 1.11 0.012 48 SEQ ID NO: 18 61888.4 2.8889 0.66 <.0001 25SEQ ID NO: 18 61889.1 4.8889 1.18 0.0033 43 SEQ ID NO: 18 61890.4 5.88891.55 0.0219 51 SEQ ID NO: 18 61891.2 7.1111 1.46 0.1417 62 SEQ ID NO: 2461895.1 4 1.02 0.0013 35 SEQ ID NO: 24 61896.1 4.75 0.68 0.0036 41 SEQID NO: 24 61896.3 6.4 1.3182 0.0418 56 SEQ ID NO: 24 61896.5 4.9 0.8360.0024 43 SEQ ID NO: 24 61896.6 3.5 0.85 <.0001 30 SEQ ID NO: 24 61897.15.2 1.96 0.033 45 SEQ ID NO: 24 61897.3 3.8 1 0.0002 33 SEQ ID NO: 2461898.1 3.2857 0.81 0.0003 29 SEQ ID NO: 17 62236.3 5.2 1.27 0.0045 45SEQ ID NO: 17 62237.2 2 0.67 <.0001 17 SEQ ID NO: 17 62237.3 3.1111 0.93<.0001 27 SEQ ID NO: 17 62239.2 3.4 1.63 0.0018 30 SEQ ID NO: 17 62240.46.1 1.83 0.0251 53

TABLE 28 Genes showing reduced plant infection to nematodes. Nematodefemale cysts per plant % of SEQ ID NO Event # Mean SE (±) p-Valuecontrol Empty vector control Mix136 5.33 0.62 1 — SEQ ID NO: 15 61433.21.17 0.31 0.0241 22 SEQ ID NO: 15 61454.2 1.83 0.75 0.1045 34 SEQ ID NO:15 61455.4 2.67 1.59 0.4177 50 SEQ ID NO: 15 61457.2 2.5 0.85 0.3301 47SEQ ID NO: 19 61535.5 2.33 0.56 0.2553 44 SEQ ID NO: 19 61536.5 2.17 0.60.1934 41 SEQ ID NO: 19 61536.6 3.67 0.72 0.9621 69 SEQ ID NO: 2662570.3 1.33 0.49 0.0358 25 SEQ ID NO: 26 62570.4 0.83 0.31 0.0104 16SEQ ID NO: 26 62570.5 0.75 0.75 0.0284 14 SEQ ID NO: 26 62571.1 0.830.54 0.0104 16 SEQ ID NO: 26 62573.5 0.17 0.17 0.0016 3 SEQ ID NO: 2662573.6 1.5 0.67 0.0522 28 SEQ ID NO: 26 62873.3 0.67 0.33 0.0066 13 SEQID NO: 26 62576.4 0.2 0.2 0.0035 4 SEQ ID NO: 27 62578.1 0.83333 0.40.0104 16 SEQ ID NO: 27 62578.3 0.8 0.8 0.0164 15 SEQ ID NO: 27 62578.40.5 0.34 0.0042 9 SEQ ID NO: 27 62579.1 1.16667 0.48 0.0241 22 SEQ IDNO: 27 62579.2 0.66667 0.33 0.0066 13

TABLE 29 Genes showing reduced plant infection to nematodes. Gene nameNematode female cysts per plant % of (SEQ ID NO) Event # Mean SE (±)p-Value control Empty vector control Mix135 8.75 2.25 1 — SEQ ID NO: 2361448.2 2.86 1 0.0003 33 SEQ ID NO: 23 61448.3 1.43 0.65 <.0001 16 SEQID NO: 23 61449.1 2.14 1.16 <.0001 24 SEQ ID NO: 23 61449.3 2.14 0.8<.0001 24 SEQ ID NO: 23 61450.1 1.67 0.49 <.0001 19 SEQ ID NO: 2361450.2 0 0 <.0001 0 SEQ ID NO: 23 61450.4 0.5 0.27 <.0001 6 SEQ ID NO:23 61452.4 1.75 0.85 0.0001 20 SEQ ID NO: 20 61724.1 0 0 <.0001 0 SEQ IDNO: 20 61724.4 0.71 0.57 <.0001 8 SEQ ID NO: 20 61725.2 3 1.2 0.0004 34SEQ ID NO: 20 61725.3 0 0 <.0001 0 SEQ ID NO: 20 61725.6 0 0 <.0001 0SEQ ID NO: 20 61726.2 2.3 0.7 <.0001 26 SEQ ID NO: 20 61727.3 1.4 0.75<.0001 16 SEQ ID NO: 22 62124.3 3.5 2.22 0.0081 40 SEQ ID NO: 22 62125.10.43 0.2 <.0001 5 SEQ ID NO: 22 62125.3 0 0 <.0001 0 SEQ ID NO: 2262126.1 1.57 0.69 <.0001 18 SEQ ID NO: 22 62126.3 4.33 1.67 0.0731 50SEQ ID NO: 22 62129.2 1 0.41 <.0001 11

Results of these experiments (Tables 26-29) indicate that the number ofcysts formed on Arabidopsis roots expressing, for example, at least onepolynucleotide having the nucleotide sequence of SEQ ID NOs:16, 20, 22,23, 24, 26, 27, 228 and/or 1047 was significantly lower than ontransgenic soybean roots comprising the empty vector control.

Similar to the polynucleotides identified as reducing nematode cystdevelopment on soybean hairy root, polynucleotides having substantialsequence identity (e.g., at least 80% identity) to the polynucleotidesshown above as reducing the number of cysts on Arabidopsis roots mayalso be useful for reducing nematode infestation, cyst number and thelike, in plants. Non-limiting examples of polynucleotides havingsubstantial identity to the nucleotide sequence of SEQ ID NO:16 includeSEQ ID NO:61 and/or SEQ ID NO:62; to the nucleotide sequence of SEQ IDNO:20 includes the nucleotide sequences of SEQ ID NO:66 and/or SEQ IDNO:67; to the nucleotide sequence of SEQ ID NO:22 includes thenucleotide sequences of SEQ ID NOs:68-112; to the nucleotide sequence ofSEQ ID NO:23 includes the nucleotide sequences of SEQ ID NOs:113-118; tothe nucleotide sequence of SEQ ID NO:24 includes SEQ ID NO:119; to thenucleotide sequence of SEQ ID NO:26 includes the nucleotide sequences ofSEQ ID NOs:120-124; to the nucleotide sequence of SEQ ID NO:27 includesthe nucleotide sequences of SEQ ID NOs:125-127; and/or to the nucleotidesequence of SEQ ID NO:228 includes the nucleotide sequences of SEQ IDNOs:399-401.

Altogether, the results from Tables 1-29 show that thepolynucleotides/polypeptides of the invention can be useful forincreasing resistance and or tolerance to plant nematodes. The foregoingis illustrative of the invention, and is not to be construed as limitingthereof. The invention is defined by the following claims, withequivalents of the claims to be included therein.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A method of controlling a nematode plant pest, comprising contacting the nematode plant pest with a transgenic plant, or part thereof, having incorporated into its genome a recombinant nucleic acid molecule that increases the expression of one or more polypeptides sequences selected from the group consisting of SEQ ID NOs: 29-42, 135-209, 243-245, 247-249, 251, 260, 665-792, 796-1018, 1020, 1025-1046, or encoding a conservatively modified variant of said polypeptide having at least 80% sequence identity to said polypeptide, thereby controlling the nematode plant pest.
 2. The method of claim 1, wherein said recombinant nucleic acid molecule is operatively linked to a promoter functional in a plant or plant cell, and wherein said nucleic acid sequence is a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-28, 43-134, 210-211, 213-215, 217-227, 229-231, 233-242, 261-398, 402-636, 638, 643-644.
 3. The method of claim 1, wherein the transgenic plant or plant part is a transgenic soybean plant, a transgenic sugar beet plant, a transgenic corn plant, a transgenic cotton plant, a transgenic canola plant, a transgenic wheat plant, or a transgenic rice plant, or a part thereof.
 4. The method of claim 1, wherein the nematode pest is selected from the group consisting of: a cyst nematode (Heterodera spp.), a root knot nematode (Meloidogyne spp.), a lance nematode (Hoplolaimus spp.), a stunt nematode (Tylenchorhynchus spp.), a spiral nematode (Helicotylenchus spp.), a lesion nematode (Pratylenchus spp.), a sting nematode (Belonoluimus spp.), a reniform nematode (Rotylenchulus reniformis), a burrowing nematode (Radopholus similis), a ring nematode (Criconema spp.), and any combination thereof.
 5. A transgenic plant, or part thereof, having incorporated into its genome a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-42, 135-209, 243-245, 247-249, 251, 260, 665-792, 796-1018, 1020, 1025-1046, or encoding a conservatively modified variant of said polypeptide having at least 80% sequence identity to said polypeptide, wherein the transgenic plant is resistant to a nematode pest.
 6. A method of producing a soybean plant having increased resistance to infestation by a nematode plant pest, the method comprising the steps of (a) crossing the transgenic plant of claim 5 with itself or another plant to produce seed comprising said recombinant nucleic acid molecule that modulates the expression of one or more polypeptides selected from the group consisting of SEQ ID NOs: 29-42, 135-209, 243-245, 247-249, 251, 260, 665-792, 796-1018, 1020, 1025-1046, and any combination thereof; and (b) growing a progeny plant from said seed to produce a plant having increased resistance to infestation by nematode plant pests.
 7. The method of claim 6, further comprising (c) crossing the progeny plant with itself or another plant and (d) repeating steps (b) and (c) for an additional 0-7 generations to produce a plant having increased resistance to infestation by nematode plant pests.
 8. A crop comprising a plurality of the transgenic plants of claim 6 planted together in an agricultural field.
 9. A method of improving yield of a plant crop, comprising: cultivating a plurality of the plants of claim 5 as a plant crop, wherein the plurality of plants of said plant crop have increased resistance to nematode infection, thereby improving the yield of said plant crop.
 10. A method of reducing the infectivity of a nematode plant pest to a plant, comprising contacting the nematode plant pest with the transgenic plant of claim 5, thereby reducing the infectivity of the nematode plant pest to the plant.
 11. A recombinant nucleic acid molecule comprising a nucleotide sequence operatively linked to a heterologous promoter functional in a plant, wherein the nucleotide sequence encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 29-42, 135-209, 243-245, 247-249, 251, 260, 665-792, 796-1018, 1020, 1025-1046, or a conservatively modified variant of said polypeptide having at least 80% sequence identity to said polypeptide.
 12. The recombinant nucleic acid molecule of claim 11, wherein said nucleotide sequence exhibits at least 80% sequence identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-28, 43-134, 210-211, 213-215, 217-227, 229-231, 233-242, 261-398, 402-636, 638, 643-644.
 13. A nematicidal composition comprising the recombinant nucleic acid molecule of claim 11 in an agriculturally acceptable carrier.
 14. The nematicidal composition of claim 13, further comprising additional nematicidal or insecticidal compounds.
 15. The nematicidal composition of claim 14, wherein the additional nematicidal compound is selected from the group consisting of chloropicrin, metam sodium, metam potassium, dazomet, iodomethane, dimethyl disulfide (DMDS), sulfryl fluoride, oxamyl and fosthiazate.
 16. A nematicidal composition comprising a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 29-42, 135-209, 243-245, 247-249, 251, 260, 665-792, 796-1018, 1020, 1025-1046, in an agriculturally acceptable carrier.
 17. The nematicidal composition of claim 16, further comprising additional nematicidal or insecticidal compounds.
 18. A method of producing a transgenic plant, the method comprising introducing into a plant a recombinant nucleic acid molecule of claim 11, thereby producing a transgenic plant having increased resistance to a nematode plant pest.
 19. The method of claim 18, wherein the introducing is done by transforming a plant cell with said recombinant nucleic acid molecule and regenerating a transgenic plant or by breeding.
 20. The method of claim 18, wherein the introducing is done by transforming a plant cell and regenerating a transgenic plant or by breeding.
 21. The method of claim 18, wherein the nematode plant pest is selected from the group consisting of: a cyst nematode (Heterodera spp.), a root knot nematode (Meloidogyne spp.), a lance nematode (Hoplolaimus spp.), a stunt nematode (Tylenchorhynchus spp.), a spiral nematode (Helicotylenchus spp.), a lesion nematode (Pratylenchus spp.), a sting nematode (Belonoluimus spp.), a reniform nematode (Rotylenchulus reniformis), a burrowing nematode (Radopholus similis), a ring nematode (Criconema spp.), and any combination thereof. 